A PP2A molecular glue overcomes RAS/MAPK inhibitor resistance in KRAS-mutant non–small cell lung cancer
Brynne Raines, Stephanie Tseng-Rogenski, Amanda C. Dowdican, Irene Peris, Matthew Hinderman, Kaitlin P. Zawacki, Kelsey Barrie, Gabrielle Hodges Onishi, Alexander M. Dymond, Tahra K. Luther, Sydney Musser, Behirda Karaj Majchrowski, J. Chad Brenner, Aqila Ahmed, Derek J. Taylor, Caitlin M. O’Connor, Goutham Narla
Brynne Raines, Stephanie Tseng-Rogenski, Amanda C. Dowdican, Irene Peris, Matthew Hinderman, Kaitlin P. Zawacki, Kelsey Barrie, Gabrielle Hodges Onishi, Alexander M. Dymond, Tahra K. Luther, Sydney Musser, Behirda Karaj Majchrowski, J. Chad Brenner, Aqila Ahmed, Derek J. Taylor, Caitlin M. O’Connor, Goutham Narla
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Research Article Cell biology Oncology

A PP2A molecular glue overcomes RAS/MAPK inhibitor resistance in KRAS-mutant non–small cell lung cancer

Abstract

The effectiveness of RAS/MAPK inhibitors in treating metastatic KRAS-mutant non–small cell lung cancer (NSCLC) is often hindered by the development of resistance driven by disrupted negative feedback mechanisms led by phosphatases like PP2A. PP2A is frequently suppressed in lung cancer to maintain elevated RAS/MAPK activity. Despite its established role in regulating oncogenic signaling, targeting PP2A with RAS/MAPK to prevent resistance has not been previously demonstrated. In this study, we aimed to establish a treatment paradigm by combining a PP2A molecular glue with a RAS/MAPK inhibitor to restore PP2A activity and counteract resistance. We demonstrated that KRASG12C and MEK1/2 inhibitors disrupted PP2A carboxymethylation and destabilized critical heterotrimeric complexes. Furthermore, genetic disruption of PP2A carboxymethylation enhanced intrinsic resistance to MEK1/2 inhibition both in vitro and in vivo. We developed RPT04402, a PP2A molecular glue that selectively stabilizes PP2A-B56α heterotrimers. In commercial cell lines and in a patient-derived model, combining RPT04402 with a RAS/MAPK inhibitor slowed proliferation and enhanced apoptosis. In mouse xenografts, this combination induced tumor regressions, extended median survival, and delayed the onset of treatment resistance. These findings highlight that promoting PP2A stabilization and RAS/MAPK inhibition presents a promising therapeutic strategy to improve treatment outcomes and overcome resistance in metastatic KRAS-mutant NSCLC.

Authors

Brynne Raines, Stephanie Tseng-Rogenski, Amanda C. Dowdican, Irene Peris, Matthew Hinderman, Kaitlin P. Zawacki, Kelsey Barrie, Gabrielle Hodges Onishi, Alexander M. Dymond, Tahra K. Luther, Sydney Musser, Behirda Karaj Majchrowski, J. Chad Brenner, Aqila Ahmed, Derek J. Taylor, Caitlin M. O’Connor, Goutham Narla

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Figure 5

Loss of B56α abolishes synergy between RPT04402 and RAS/MAPK inhibition.

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Loss of B56α abolishes synergy between RPT04402 and RAS/MAPK inhibition....
(A) 2D contour plots of highest single-agent synergy scores for B56α–/– cell lines (A549, NCI-H358) cultured as 2D monolayers or 3D spheroids. Cells were treated with increasing concentrations of RPT04402, trametinib (A549), or adagrasib (NCI-H358), either as single treatments or in combination, for 48 hours. (B) Representative images of 2D and 3D live/dead staining in A549 (trametinib: 2D/3D = 30 nM; RPT04402: 2D/3D = 25 μM) and NCI-H358 (adagrasib: 2D/3D = 150/3 nM; RPT04402: 2D/3D = 22.5 μM) cells after 48 hours of treatment. Scale bar: 75 μm. (C) Clonogenic assay of A549 cells treated with increasing concentrations of trametinib, RPT04402, or the combination, cultured for 2 weeks. (D) Clonogenic assay of NCI-H358 cells treated with increasing concentrations of adagrasib, RPT04402, or the combination, and cultured for 2 weeks. (E) Western blot analysis of pERK, mePP2ACα, and PP2ACα in A549 EV and B56α–/– cells after 48 hours of treatment with trametinib (30 nM), RPT04402 (25 μM), and the combination. (F) Western blot analysis of pERK, mePP2ACα, and PP2ACα in NCI-H358 EV and B56α–/– cells after 48 hours of treatment with adagrasib (150 nM), RPT04402 (27.5 μM), and the combination. For both E and F, pERK is normalized to tERK and mePP2ACα, and PP2ACα is normalized to the loading control, vinculin. Densitometry data are expressed relative to the DMSO condition within the genetic background. Vinculin blots are in Supplemental Figure 13. Data in graphs are represented as mean ± SEM (n ≥ 3). Statistical significance was determined by 1-way ANOVA with Tukey’s post hoc analysis: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Raw values are in the Supporting Data Values file.