RAS mutation-specific signaling dynamics in response to paralog- and state- selective RAS inhibitors

A high therapeutic index (TI), balancing potent oncogenic signaling inhibition in tumor cells with minimal effects on normal cells, is critical for effective cancer therapies. Recent advances have introduced diverse RAS-targeting inhibitors, including mutant-specific inhibitors (e.g., KRAS(G12C) and KRAS(G12D)), as well as paralog- and state-selective inhibitors. Non-mutant-specific RAS inhibition can be accomplished by 1) panRAS-GEF(OFF) inhibitors which inactivate RAS indirectly by inhibiting SHP2 or SOS1, thereby blocking the nucleotide exchange step of RAS activation, 2) direct KRAS(OFF)-selective inhibitors sparing NRAS and HRAS, and 3) panRAS(ON) inhibitors that directly target active RAS, by occluding binding of its effector RAF. However, the signaling inhibition index (SII) - the differential inhibition of oncogenic signaling between RAS-mutant (RAS(MUT)) and normal cells - remains poorly defined for these approaches. In this study, we evaluated the SII of state- and paralog-selective RAS inhibitors across diverse RAS-mutant (RAS(MUT)) and RAS-wild-type (RAS(WT)) models. PanRAS-GEF(OFF) inhibitors exhibited neutral or negative SII, with comparable or reduced MAPK suppression in KRAS(G12X) cells relative to RAS(WT) cells. KRAS(G13D) models showed low sensitivity (negative SII) to panRAS-GEF(OFF) inhibitors, particularly in the context of NF1 loss. Combination treatments with SHP2 and MEK inhibitors resulted in low SII, as pathway suppression was similar in RAS(MUT) and RAS(WT) cells. Furthermore, RAS(Q61X) models were resistant to combined SHP2 inhibitor+MEK inhibitor due to dual mechanisms: MEK inhibitor-induced NRAS(Q61X) reactivation and RAS(MUT)-induced SHP2 conformations impairing inhibitor binding. Overall, panRAS-GEF(OFF) inhibitors exhibited the lowest SII. PanKRAS(OFF) inhibitors demonstrated a higher SII, while panRAS(ON) inhibitors displayed broader activity but relatively narrow SII. We observed that tumors that were sensitive to RAS(MUT)-specific inhibitors, were also sensitive to the state-selective RAS inhibitors (OFF, or ON). In fact, all RAS inhibitors (mutant-specific and state- or paralog-selective) were active in the same portion of RAS(MUT) models, while the majority of RAS(MUT) cell lines were insensitive to all of them. These findings reveal significant SII variability among RAS-targeted inhibitors, depending on the specific RAS driver mutation and cell context and underscore the importance of incorporating SII considerations into the design and clinical application of RAS-targeted therapies to improve therapeutic outcomes. PanRAS-GEF(OFF) inhibitors have limited SII and effectiveness: The Signaling Inhibition Index (SII) - i.e. the differential inhibition of oncogenic signaling between tumor and normal cells - was neutral or negative for panRAS-GEF(OFF) inhibitors, with comparable or reduced MAPK suppression in KRAS(G12X) mutant versus RAS(WT) cells. KRAS(G13D) models showed reduced sensitivity, particularly with NF1 loss. SHP2+MEK inhibitor combinations also had low SII, with RAS(Q61X) models demonstrating resistance due to NRAS(Q61X) reactivation and impaired SHP2 inhibitor binding.PanKRAS(OFF) selective inhibitors have higher SII than panRAS-GEF(OFF) inhibitors: panKRAS(OFF)-selective inhibitors have a higher SII compared to panRAS-GEF(OFF) inhibitors, offering better tumor-versus-normal cell selectivity.PanRAS(ON) inhibitors have broad but modest SII: While panRAS(ON) inhibitors displayed a broader activity profile, their ability to selectively inhibit mutant RAS signaling over normal cells remained relatively narrow (low SII).Most KRAS-mutant tumors will be insensitive to any single RAS-targeted inhibitor: State- and paralog-selective inhibitors have enhanced activity in the same RAS-MUT cancer models that are also sensitive to RAS-MUT-specific inhibitors, suggesting that most KRAS-MUT tumors will not respond uniformly to any one RAS-targeting inhibitor.SII varies across RAS inhibitors, necessitating tailored therapeutic strategies: The effectiveness of paralog- and state-selective inhibitors depends on specific RAS mutations and cell context, highlighting the need to integrate SII considerations into the development and clinical application of RAS-targeted therapies.