Abstract 1827: Direct activation of the tumor suppressor protein phosphatase 2A as a therapeutic strategy for TKI-resistant lung adenocarcinoma
Authors: Tohme Rita , Sangodkar Jaya , Dhawan Neil , Izadmeher Sudeh , Sharma Neelesh , Ohlmeyer Michael , Narla Goutham 
Published: August 2018

Most lung adenocarcinoma (LUAD) patients develop acquired resistance to tyrosine kinase inhibitors (TKI) via mechanisms enabling the sustained activation of the MAPK and PI3K oncogenic pathways downstream of the tyrosine kinase 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 MAPK and AKT pathways and is a promising therapeutic strategy for TKI-resistant LUAD. LUAD cell lines A549, H1975, and H1650 modeling intrinsic and acquired modes of TKI-resistance were treated with Small Molecule Activator of PP2A (SMAP) developed in our lab. RNAseq canonical pathway analysis revealed that PP2A activation resulted in significant upregulation in major apoptotic cell death pathways and downregulation in growth and proliferation pathways, namely the canonical PI3K and MAPK signaling pathways. Kinase enrichment analysis followed by principal component analysis indicated that SMAP treatment induces a gene signature similar to a combination of the selective AKT and MEK inhibitors MK2206 and AZD6244, respectively. Cell-cycle arrest, caspase-dependent apoptosis, and reduced colony formation ability were observed in TKI-resistant cells. SMAP treatment caused a dephosphorylation of AKT and ERK resulting in downregulation of the AKT and MAPK pathways in H1650. A549 and H1975, with lower baseline pAKT levels, experienced a significant dephosphorylation of ERK. These results suggest that the dephosphorylation effect mediated by PP2A activation is highly dependent on the availability of the phosphatase's target. Indeed, stably overexpressing myristoylated AKT in H1975 led to a dual-pathway inhibition upon SMAP treatment. The therapeutic potential of PP2A activation in vivo was first evaluated in a transgenic EGFRL858R mouse model harboring an activating transgene directing the expression of mutant EGFR to the Clara cells. The reticulonodular pattern observed with magnetic resonance imaging was recapitulated by hematoxylin and eosin staining of lung samples, where mice treated with SMAP showed less diffuse lung cancer and a significant decrease in total nodules. Immunohistochemistry revealed increased TUNEL staining, decreased PCNA staining, and dephosphorylation of both ERK and AKT. Single-agent SMAP demonstrated significant tumor growth inhibition, as well as ERK and AKT dephosphorylation, in a TKI-resistant patient-derived xenograft model, in a comparable fashion to a combination of AZD6244 and MK2206 kinase inhibitors. Combination of SMAP and the TKI Afatinib resulted in an enhanced effect on cell death and tumor growth inhibition in a H1975 xenograft model. SMAP treatment was well tolerated and had no notable toxicities in vivo. These collective data support the development of PP2A activators for the treatment of TKI-resistant LUAD.

Citation Format: Rita Tohme, Jaya Sangodkar, Neil Dhawan, Sudeh Izadmeher, Neelesh Sharma, Michael Ohlmeyer, Goutham Narla. Direct activation of the tumor suppressor protein phosphatase 2A as a therapeutic strategy for TKI-resistant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1827.