KRAS-mutant non-small cell lung cancer (NSCLC) remains one of the most difficult cancers to treat long-term. While targeted therapies such as MEK inhibitors or, more recently, KRAS-G12C inhibitors have transformed and advanced the treatment landscape, the benefits seen in patients are often short-lived as resistance emerges almost universally.
Resistance to RAS/MAPK targeted therapies is driven by both intrinsic and acquired mechanisms. During acquired resistance, tumors rapidly adapt by rewiring signaling cascades, specifically MAPK, to evade pathway inhibition and dampen therapeutic response. Therefore, discovering and functionally validating the role of these signaling networks is paramount to designing rational treatment strategies to improve response and patient outcomes.
Targeted therapies have largely been directed at kinases, with RAS/MAPK inhibitors designed to suppress MAPK signaling by blocking kinase mediated phosphorylation. However, phosphorylation is a reversible process, and its reversal is driven and highly regulated by protein phosphatases. Thus, the activity of oncogenic K-Ras is not solely defined by how strongly it drives MAPK signaling, but also by how weakly phosphatases counteract this signaling network. In normal cells, kinases and phosphatases exist in a tightly regulated equilibrium, and it is only when this balance is disrupted that cancer thrives.
In our newly published study in The Journal of Clinical Investigation (JCI), we have discovered a contributor to the resistance of RAS/MAPK inhibitors: specifically the tumor's dependence on disrupting the natural balance between kinases and phosphatases, in particular the serine/threonine phosphatase, Protein Phosphatase 2A (PP2A).
PP2A describes a family of serine/threonine phosphatases that function as heterotrimeric complexes composed of a scaffolding, catalytic, and regulatory subunit, with the latter conferring substrate specificity. Specific PP2A heterotrimers act as tumor suppressors and their inhibition is required for Ras driven cellular transformation. Importantly, PP2A negatively regulates K-Ras mediated MAPK signaling and hyperactivation of this pathway is a well-established mechanism of resistance.
We observed that treating KRAS-mutant NSCLC cells with a MEK or KRAS-G12C inhibitor induced the loss of a key post-translational modification of PP2A, carboxymethylation. Reversible carboxymethylation at the catalytic subunit drives PP2A heterotrimer biogenesis and determines whether PP2A is tumor-promoting or tumor-suppressive based on which regulatory subunit binds. When we treated cells with a MEK or KRAS-G12C inhibitor, the loss of methylation drove the specific loss of the tumor-suppressive holoenzymes, specifically the PP2A-B56α heterotrimer. We also observed that genetic ablation of carboxymethylation drove intrinsic MEK inhibitor resistance in KRAS mutant in vivo models of the disease further underscoring the importance of this post-translational modification in driving broad MAPK inhibitor resistance. Collectively, our data suggests that the loss of carboxymethylation and destabilization of specific PP2A tumor suppressive heterotrimers allowed cells/tumors to reactivate MAPK signaling and bypass therapeutic blockage.
Therapeutically, we demonstrated that the pharmacological reactivation of PP2A using a next-generation PP2A molecular glue, RPT04402, restabilized PP2A-B56α heterotrimers. When we combined this first-in-class PP2A molecular glue, RPT04402 with RAS/MAPK inhibitors in cell-based studies, we observed sustained ERK suppression, increased apoptosis, drug synergy across multiple cell lines, and the collapse of long-term colony outgrowth (mimicking in vivo studies). These data indicated that KRAS-mutant NSCLC cells require phosphatase suppression to escape MAPK inhibition and that restoring this activity eliminates escape.
Most importantly, our cell-based studies were replicated in vivo. In xenograft models treated over 150 days, RPT04402-potentiated RAS/MAPK inhibitor response blocked the development of acquired resistance compared to single-agent treatment arms. Tumors displayed suppressed ERK phosphorylation and enhanced apoptotic signaling in addition to sustained tumor growth inhibition.
These results highlight the therapeutic utility of restoring the signaling balance rather than targeting kinases alone and call for a shift in how we view resistance to RAS/MAPK targeted therapies. Resistance results not only from increased kinase activity, but also from decreasing phosphatase activity. This shift in view has several implications: phosphatases are not passive bystanders but active regulators of therapeutic response, adaptive resistance is a two-sided equation, and targeting only the kinase arm of these signaling networks may be insufficient to drive deep and durable therapeutic responses. Our results demonstrate that reactivating PP2A restores the baseline architecture of signaling, and future therapies should consider both sides of the kinase-phosphatase balance.
Ultimately, we envision a future where regulating phosphatase activity becomes a therapeutic strategy, used alongside kinase inhibitors to produce deeper and more durable responses in KRAS-mutant cancers.
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