Phosphoproteomics Profiling of Nonsmall Cell Lung Cancer Cells Treated with a Novel Phosphatase Activator

Abstract

Activation of protein phosphatase 2A (PP2A) is a promising anticancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems-level perturbations mediated by PP2A activation, we carried out mass spectrometry-based phosphoproteomic analysis of two KRAS mutated non-small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel small molecule activator of PP2A (SMAP). Overall, this permitted quantification of differential signaling across over 1600 phosphoproteins and 3000 phosphosites. Kinase activity assessment and pathway enrichment implicate collective downregulation of RAS and cell cycle kinases in the case of both cell lines upon PP2A activation. However, the effects on RAS-related signaling are attenuated for A549 compared to H358, while the effects on cell cycle-related kinases are noticeably more prominent in A549. Network-based analyses and validation experiments confirm these detailed differences in signaling. These studies reveal the power of phosphoproteomics studies, coupled to computational systems biology, to elucidate global patterns of phosphatase activation and understand the variations in response to PP2A activation across genetically similar NSCLC cell lines.

PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells

“…S4, B and C), suggesting that these cells are already saturated for CIP2A-mediated PP2A inhibition. Finally, in the context of mTOR/AKT signaling as the mechanism of MEKi resistance resulting from PP2A inhibition, SMAP DT-061 inhibits AKT and RPS6K signaling in H358 cells (38). We confirmed that the DT-061 + MEKi combination potently inhibited the identified MEKi resistance mechanism (probed by p-AKT and MYC antibodies) in KRAS-mutant lung cancer cell lines H358 and H441 ( fig.…”

RABL6A inhibits tumor-suppressive PP2A/AKT signaling to drive pancreatic neuroendocrine tumor growth

“…These tricyclic compounds were originally derived from neuroleptic drugs that were reengineered to neutralize their CNS effects, resulting in a novel class of specific PP2A-activating compounds (51,52). Through extensive testing, SMAPs have shown remarkable efficacy and specificity in stimulating PP2A phosphatase activity toward oncogenic targets (such as phosphorylated ERK1/2) and halting the growth of lung and prostate cancer cells, both in vitro and in vivo, in a PP2A-dependent manner (53)(54)(55)(56). We transiently treated parental BON-1 cells, as well as Qgp1 cells (the only other PNET cell line available at the time of this study), overnight with increasing doses of SMAP.…”

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

“…PP2A activation by SMAPs simultaneously inhibits MAPK and PI3K oncogenic signaling pathways, which are downstream targets of the phosphatase, and their dephosphorylation seems to be regulated by the cellular abundance of target proteins ERK and AKT. We have shown in our recently published work that direct therapeutic activation of a phosphatase, such as PP2A, is a potentially novel strategy that aims to concurrently target several oncogenes with a single agent (16)(17)(18). Indeed, we have found that SMAPs directly bind to and activate PP2A in KRAS-mutant LUAD, therefore inhibiting tumor growth and inducing apoptosis in mouse xenografts, PDX models, and the transgenic KRAS LA2 murine model (17).…”

“…Our lab has successfully developed a published series of first-in-class small molecule activators of PP2A (SMAPs), which directly bind to PP2A and induce apoptosis and tumor growth inhibition in both transgenic and xenograft KRAS-driven mice models (16)(17)(18). SMAPs have been generated by repurposing and reengineering FDA-approved tricyclic neuroleptics through replacing the basic amine with a neutral polar functional group (19).…”