Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).


RMC-7977 discovery and development
RAS proteins have historically been recalcitrant drug targets 2,3 , although progress in targeting the inactive, GDP-bound state of KRAS(G12C) has resulted in regulatory approvals for two drugs, sotorasib and adagrasib 10,11 .We recently described RMC-4998 and RMC-6291 12 , two covalent tri-complex inhibitors that are designed to target the active state of KRAS(G12C).These macrocyclic compounds were derived from sanglifehrin A, a natural product that binds to cyclophilin A (CYPA) with high affinity 13 .The mechanism of action of these inhibitors is distinct from that of bifunctional immunophilin-binding inhibitors with independent RAS-and CYPA-binding motifs joined by a linker 14 , and instead reflects the binding mechanism of multiple natural products that inspired a paradigm for inhibiting undruggable targets 15,16 .Upon binding CYPA, tri-complex inhibitors remodel the surface of CYPA to create a binary complex with high affinity for active KRAS.Selectivity for KRAS(G12C) is achieved via covalent modification of the reactive thiol group introduced by the oncogenic mutation.The resulting CYPA-compound-KRAS tri-complex sterically blocks KRAS-effector interactions and disrupts downstream signalling.
Most RAS oncoproteins with missense mutations are not amenable to selective covalent targeting but could be susceptible to non-covalent inhibition by tri-complex formation with CYPA.In a previous study, we identified compound 2 12 (referred to in the present Article as compound 1) (Fig. 1a) with weak, reversible binding to GMPPNP-bound wild-type KRAS and KRAS(G12C).We postulated that we could use structure-guided design to optimize compound 1 to generate a reversible, orally bioavailable inhibitor with broad activity against the active states of multiple RAS variants.Tri-complex formation requires two distinct binding events (Fig. 1b).First, the compound binds to CYPA to form the binary complex (with dissociation constant K d1 ).The binary CYPA-compound complex then binds to active RAS (with dissociation constant K d2 ) to form a tri-complex structure in which CYPA sterically occludes RAS-effector interactions (Extended Data Fig. 2a-d).Both binding events are essential for tri-complex formation, and we sought to optimize both K d1 and K d2 to increase the potency of RAS inhibition, focusing initially on KRAS(G12V) as a representative oncogenic mutant.
A high-resolution co-crystal structure of RMC-7977 bound to CYPA and GMPPNP-bound KRAS shows a tri-complex with extensive noncovalent interactions and an unoccupied groove containing the common oncogenic residues, G12, G13 and Q61, providing a structural basis for the ability of RMC-7977 to bind these variants (Fig. 1f,g, PDB IDs: 8TBF, 8TBH, 8TBL and 8TBM and unpublished data).Further, RMC-7977 exhibited a consistent binding mode across all KRAS(G12X) mutants tested (Extended Data Fig. 2j, PDB IDs: 8TBF, 8TBG, 8TBH, 8TBI, 8TBJ, 8TBK, 8TBL, 8TBM and 8TBN).K d2 values for the most common oncogenic RAS variants were all within threefold of those for wild-type proteins (Extended Data Table 2).The ability of tri-complex formation to sterically disrupt effector binding for the various mutants was also measured, revealing a good correlation between the K d2 measurements and the biochemical EC 50 values for RAS-RAF disruption (Fig. 1h).Similar potency was also observed for inhibition of KRAS(G12V)-RALGDS (RAS-interacting domain (RID)) binding in vitro (Extended Data Fig. 3a).Coincubation with increasing concentrations of BRAF RBD attenuated tri-complex formation, indicating it is competitive with effector binding (Extended Data Fig. 2l).
We used a live-cell nano-bioluminescence resonance energy transfer (BRET) kinetic assay to show that RMC-7977 induced equally rapid (signal half-life (t 1/2 ) < 5 min; Fig. 2a) association between KRAS and CYPA and dissociation of the CRAF RBD from KRAS, consistent with direct targeting of the active state of RAS in cells accompanied by steric inhibition of protein-protein effector engagement.EC 50 measurements in this assay were in the single-digit nanomolar range across a panel of wild-type, G12, G13 and Q61-mutant KRAS proteins, and correlated well with EC 50 values for induced KRAS-CYPA association (Fig. 2b).Similar potencies were observed for inhibition of RALGDS, PI3Kα and SOS1 binding to KRAS(G12C), KRAS(G12V) or KRAS(G12D), as well as SOS1 binding to wild-type KRAS (Extended Data Fig. 3b-d).Tri-complex formation induced by RMC-7977 was also more than tenfold more potent for KRAS than MRAS and other RAS family small GTPase proteins with high sequence identity to KRAS (Extended Data Fig. 3e,f).
The cellular potencies for KRAS-CYPA association were approximately 5 to 50 times higher than the corresponding biochemical K d2 measurements (Extended Data Tables 1 and 2).An increase in cellular potency compared with biochemical potency is expected based on the tri-complex mechanism of action, in which binding to abundant CYPA drives high intracellular concentrations of CYPA-RMC-7977 binary complexes, as evidenced by accumulation of RMC-7977 in a CYPA-dependent manner in AsPC-1 cells (Extended Data Fig. 4a).Furthermore, biochemical and cellular potencies are similar when adjusted to reflect the estimated intracellular concentration of binary complexes formed in cells (Extended Data Fig. 4b,c).
To verify that formation of the CYPA-RMC-7977 binary complex is essential for cellular activity, we used a competitive CYPA inhibitor 17 or genetically knocked out PPIA, the gene that encodes CYPA.1-3).

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These studies confirm that CYPA binding is required for inhibition of RAF-MEK-ERK signalling and proliferation by RMC-7977 in NCI-H441 (KRAS G12V , NSCLC) and AsPC-1 (KRAS G12D , PDAC) cells (Extended Data Fig. 4d-g).As a control, disruption of the PPIA locus did not affect sensitivity to the MEK1/2-selective inhibitor, trametinib, which does not rely on the tri-complex mechanism of action (Extended Data Fig. 4h,i).We further investigated whether exogenous CYPA expression could restore RMC-7977 sensitivity in NCI-H358 (KRAS G12C , NSCLC) cells lacking PPIA.We investigated two clones expressing either low or high CYPA levels through a doxycycline-inducible promoter (Extended Data Fig. 5a).Inhibition of pERK and proliferation was CYPA-dependent, and CYPA-high cells were threefold and eightfold more sensitive to RMC-7977 inhibition of signalling and cell proliferation, respectively, compared with CYPA-low cells (Fig. 2c and Extended Data Fig. 5b).RMC-7977 accumulation was significantly greater in CYPA-high cells compared with CYPA-low cells treated with 10 nM RMC-7977, with no difference at 1 µM, at which concentration binding to cellular CYPA is estimated to approach saturation (Fig. 2d).Collectively, these observations suggest that the cellular potency of RMC-7977 is dependent on intracellular concentration of binary complexes, driven by intracellular CYPA protein expression.CYPA is highly abundant in cells (median concentration = 12.3 µM) as measured across a panel of 15 cell lines (Extended Data Fig. 5c), and CYPA expression was higher in cell line-derived xenograft (CDX) tumours in vivo compared with the corresponding cells cultured in vitro (Extended Data Fig. 5d).Finally, CYPA is abundantly expressed across cancer types and exhibits low inter-patient variation in expression 12 , suggesting that tumour expression of CYPA is unlikely to be limiting for RMC-7977 potency.Indeed, PPIA mRNA expression across a panel of cancer cells did not correlate with sensitivity to RMC-7977 (Extended Data Fig. 5e).
RMC-7977 exhibited similar activity for wild-type and mutant RAS variants in biochemical assays, and in the live-cell nano-BRET assay the cellular potency for inhibition of CRAF (RBD) binding to wild-type KRAS was only modestly lower than that for the oncogenic variants.However, many factors can influence the downstream consequences of RAS inhibition in cells.To assess the spectrum of RMC-7977 activity against common KRAS variants in cells, we evaluated a panel of matched mouse embryonic fibroblasts (MEFs) that were null for all three Ras genes (RAS-less), in which proliferation was restored with ectopic expression of wild-type or mutationally activated 18 KRAS or BRAF V600E (Fig. 2e).RMC-7977 suppressed pERK in all KRAS-expressing cells, but not in BRAF(V600E)-expressing RAS-less MEFs, which lack all RAS proteins and are not RAS-dependent, indicating that pERK suppression is KRAS-dependent.Notably, we observed minor but consistent differences between the various KRAS mutants.pERK suppression by RMC-7977 typically appeared complete across cells expressing various KRAS(G12X) mutants, but consistently reached a plateau in wild-type KRAS, KRAS G12A , KRAS Q61H , KRAS Q61R , KRAS G13D and KRAS A146T cells; by contrast and as expected, trametinib reduced pERK similarly in all cells, including the BRAF V600E MEFs (Extended Data Fig. 6a).These differences indicate that KRAS genotype could affect the cellular response to direct RAS inhibition, and that the cellular response to RMC-7977 inhibition is not equivalent to that of MEK inhibition.
We also compared RMC-7977 activity in cancer cells harbouring various activating mutations in the RAS pathway, specifically oncogenic variants of KRAS, NRAS, EGFR or BRAF.RAS-dependent (KRAS, NRAS or EGFR-mutated) cancer cells treated with RMC-7977 exhibited concentration-dependent inhibition of downstream signalling and proliferation in the low nanomolar range (Fig. 2f,g).In KRAS G12V and KRAS G12C cells, inhibition of additional markers, including phosphorylation of RAF, ERK and the ERK substrate RSK, was demonstrated (Extended Data Fig. 6b).In these cells, there was evidence of durable pathway suppression and apoptosis induction after 48 h of treatment, indicated by sustained pERK, pCRAF and pRSK suppression and moderately increased PARP cleavage (Extended Data Fig. 6b,c).No inhibition by RMC-7977 was observed in RAS-independent BRAF V600E -mutant A375 cells (Fig. 2f,g).

RMC-7977 activity in RAS-addicted cancer
We next performed a cell viability assay in 869 human tumour cell lines of different genetic and histological subtypes in a pooled, multiplexed format (PRISM assay) to identify genetic features associated with RMC-7977 sensitivity or resistance.Oncogenic KRAS mutation status provided the most significant genetic marker of sensitivity to RMC-7977 (Fig. 3a).Similar results were observed for NRAS mutations, although no correlation with HRAS mutation status was detected, owing to the low representation of HRAS mutations (HRAS-mutated n = 22; Extended Data Fig. 7a,b).Unsurprisingly, among cell lines with BRAF mutations, BRAF class I V600 mutations were the most abundantly represented and clearly associated with resistance, as BRAF is a kinase effector of RAS and V600 mutations render BRAF RAS-independent.Cell lines with less common class II or III mutations, which remain somewhat dependent on upstream RAS signalling and frequently co-occur with RAS mutations, were often sensitive to RMC-7977, as were many unclassified BRAF mutations (Extended Data Fig. 7c).

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are frequently co-mutated with NF1 or receptor tyrosine kinase (RTK) genes, which may affect RAS dependency and, by extension, RMC-7977 sensitivity 24 .Several KRAS wild-type genotypes, including NRAS and HRAS mutant cell lines (median EC 50 = 6.76 nM), and cell lines with mutationally activated RTKs also responded to RMC-7977 inhibition, including those with mutations or fusions of EGFR, ERBB3, FGFR1, FGFR2, FGFR3, ROS1, RET, NTRK1 and ALK (median EC 50 = 6.14 nM), and cell lines with wild-type MET gene amplification (median EC 50 = 6.61 nM; Extended Data Fig. 7f).Cell lines with NF1 loss of function and PTPN11 mutations, which each cause activation of wild-type RAS signalling, were moderately sensitive (median EC 50 = 28.1 nM).Together, these data are concordant with our genetic analysis of RAS dependence and support the on-target pharmacological activity of RMC-7977.
We then assessed the pharmacodynamic and anti-tumour activity of RMC-7977 in vivo in the NCI-H441 CDX model of NSCLC (KRAS G12V , NSCLC).The relationship between the total tumour concentration of RMC-7977 and inhibition of the RAS pathway transcriptional target DUSP6 in tumour lysates yielded an EC 50 of 130 nM (Extended Data Fig. 8a), consistent with the measured KRAS(G12V) K d2 of 85 nM (Extended Data Table 1), and with the model for tri-complex RAS inhibition discussed above.A single oral dose of 10 mg kg −1 RMC-7977 was sufficient to maximally suppress tumour DUSP6 levels (91%) at 8 h, which partially recovered over 48 h, concordant with the decrease in tumour RMC-7977 concentrations (Fig. 3c).Prolonged RMC-7977 exposure in tumours was observed in this and other subcutaneously implanted xenograft tumour models, resulting in an approximately threefold increase in overall exposure in subcutaneous tumours compared with blood (Extended Data Fig. 8b).Repeated daily administration of RMC-7977 at 10 mg kg −1 was well tolerated and resulted in 83% mean tumour regression following 28 days of treatment in the NCI-H441 model (Fig. 3d).
RMC-7977 caused tumour growth inhibition and induced multiple tumour regressions across a larger panel of 15 PDAC, CRC and NSCLC CDX and patient-derived xenograft (PDX) models bearing KRAS G12X mutations and co-mutations representative of the genomic landscape of patients with KRAS-mutant cancers (Fig. 3e).RMC-7977 treatment resulted in mean tumour regression in 9 out of 15 (60%) models after a 4-to 6-week treatment period (Fig. 3e) and had a minimal effect on body weights in all models (Extended Data Fig. 8c).Of note, when we continued RMC-7977 treatment in these xenograft models for up to 90 days, the anti-tumour activity of RMC-7977 was found to remain durable as the majority of regressions and even cytostatic responses were maintained.Whereas the controls exhibited a short median time to tumour doubling of 7 days, RMC-7977 treated tumours did not reach a median time to tumour doubling (defined as tumour progression) on treatment in a Kaplan-Meier analysis of these results (Fig. 3f; Cox proportional hazard ratio 0.004, 95% interval 0.0011-0.0191,P < 1 × 10 −12 ).
MEK and ERK inhibitors have undergone extensive clinical testing as monotherapies or in combinations with other RAS pathway inhibitors in patients with KRAS or NRAS mutated cancers 25 .Despite encouraging preclinical results, these therapeutic strategies have so far been unsuccessful in the clinic [26][27][28] , with therapeutic benefits probably compromised by dose-limiting toxicities [29][30][31] .We compared the anti-tumour activity of single agent RMC-7977 to that of the upstream and downstream RAS-MAPK pathway inhibitors RMC-4550 (SHP2 inhibitor) and cobimetinib (MEK inhibitor), respectively, administered as single agents or in combination, in three KRAS G12X models.At well-tolerated doses, RMC-7977 induced deep regressions in all three models.By contrast, following administration of MEK and SHP2 inhibitors at doses that were well-tolerated and translatable, either alone or in combination, only modest tumour growth inhibition was observed (Fig. 3g).These data demonstrate that in these preclinical models of KRAS G12X mutant cancers, direct targeting of active RAS with RMC-7977 elicits a differentiated and superior anti-tumour activity profile compared with upstream and/or downstream vertical inhibition of the oncogenic driver.
There are several potential explanations for why RMC-7977 elicits greater anti-tumour activity in KRAS G12X -driven cancers compared with agents that target upstream or downstream nodes on the RAS pathway.These include more efficient suppression of oncogenic RAS signalling, relatively less impact on normal tissues 32 , or a combination of both.Directly targeting the RAS oncoprotein itself may exploit the high degree of oncogene addiction of KRAS G12X (and NRAS)-mutated cancer cells to a greater degree than targeting upstream and downstream signalling proteins (such as SHP2, MEK1/2 and ERK1/2).Furthermore, whereas MEK inhibition did not distinguish between wild-type and mutant RAS variants (Extended Data Fig. 6a), RMC-7977 exhibited modestly lower potency and incomplete wild-type RAS suppression compared with KRAS(G12X) in cells (Fig. 2b,e and Extended Data Table 2).Additionally, the slow elimination of RMC-7977 observed in subcutaneous xenograft tumours relative to blood (Fig. 3c and Extended Data Fig. 8b) suggests that it is differentially distributed to tumours, which may contribute to a wider therapeutic index.Of note, PPIA mRNA expression is reportedly induced by hypoxia under control of HIF1A and has a critical role in tumorigenesis 33,34 .Consistent with these reports, subcutaneous xenograft tumours express increased amounts of CYPA protein compared with cells grown in vitro under normoxic conditions (Extended Data Fig. 5d) and PPIA mRNA expression is increased in tumour cells 35 .Collectively, these data support the notion that CYPA is critical for tumour maintenance and may also affect tumour distribution and cellular retention of tri-complex inhibitors.
We interrogated the potential for RMC-7977-mediated inhibition of wild-type RAS to impair immune cell function in both naive and tumour-bearing immunocompetent mice.Tumour-naive mice were able to mount a CD8 + T cell response to ovalbumin peptide vaccination in the presence of RMC-7977 treatment (Extended Data Fig. 9a,b).Furthermore, RMC-7977 increased tumour-antigen-specific CD8 + T cell infiltration into KRAS G12C syngeneic tumours (Extended Data Fig. 9c-e).

Overcoming KRAS(G12C) OFF resistance
Although inactive-state KRAS(G12C) inhibitors provide short-term therapeutic benefit for some patients, most eventually relapse through acquired genetic or adaptive mechanisms of resistance [36][37][38][39] .Ryan et al. 39 reported that adaptive feedback reactivation of upstream RTK signalling through wild-type RAS limits the activity of KRAS(G12C) inhibitors.We observed analogous results in KRAS(G12D) mutant PDAC cell lines treated with the KRAS(G12D)-selective inhibitor, MRTX1133, in which pERK suppression seen at 2 h rebounded by 48 h after treatment (Fig. 4a).We hypothesized that RMC-7977 could address adaptive RAS signalling mechanisms that rely on increased active-state wild-type and mutant RAS proteins.Consistent with this hypothesis, RMC-7977 showed sustained pERK suppression in KRAS G12D PDAC cells in culture for 48 h, suggesting that broad inhibition of RAS family proteins can overcome the adaptive feedback observed with mutant-selective inhibitor treatment (Fig. 4a).Similar sustained pERK suppression and moderate PARP cleavage were also observed in two additional KRAS-mutant cancer cells (Extended Data Fig. 6b,c).We therefore hypothesized that the concurrent suppression of wild-type and mutant RAS signalling could drive durable anti-tumour responses to RMC-7977 treatment in vivo.As described above, a 90-day treatment study in a series of KRAS(G12X) xenograft models demonstrated a marked and significant increase in time to tumour doubling from baseline compared with controls (Fig. 3f).
The activity of RMC-7977 against multiple forms of oncogenic RAS suggests the potential for therapeutic benefit against resistance mechanisms involving secondary RAS mutations.Tri-complex KRAS(G12C) (ON) inhibitors, such as RMC-4998, bind to RAS through a unique mechanism and a binding site distinct from the switch II pocket occupied by inactive-state KRAS(G12C) inhibitors, such as adagrasib and sotorasib 12,[40][41][42] .Switch II pocket binding mutations such as those at positions R68, Y96 and H95 had little or no effect on RMC-7977 potency (Extended Data Fig. 10a), as was observed for RMC-4998 (ref.36).Next, we tested whether the broad-spectrum RAS inhibitory activity of RMC-7977 could counter additional genetic resistance mechanisms observed in relapsed patients treated with KRAS(G12C) inhibitors, including secondary oncogenic RAS mutations and RTK activation.Indeed, RMC-7977 inhibited RAS signalling and growth of a NCI-H358 (KRAS G12C , NSCLC) clone with a concurrent NRAS Q61K mutation that emerged in cells grown under continuous exposure to adagrasib in vitro (Fig. 4b,c and Extended Data Fig. 10b).RTK amplification and activating mutations can also cause RAS pathway reactivation through mutant and wild-type RAS proteins.We used an engineered system with doxycycline-inducible constructs of full-length and fusion RTKs previously detected in patients who progressed on adagrasib or sotorasib treatment 36 .Overexpression of wild-type or mutant RTKs in NCI-H358 cells (KRAS G12C , NSCLC) conferred reduced sensitivity to adagrasib (proliferation inhibition EC 50 shift: wild-type EGFR, 42-fold; EGFR A289 , 153-fold; HER, 51-fold; FGFR, 18-fold, RET M918 , 34-fold), but not to RMC-7977 (proliferation inhibition EC 50 shift ≤ 3-fold) (Fig. 4d,e).A similar trend was seen for inhibition of pERK inhibition (Extended Data Fig. 10c,d).Similar results were observed when oncogenic RTK fusion proteins (EML4-ALK, FGFR3-TACC3 and CCDC6-RET) were exogenously expressed in MIA PaCa-2 cells (KRAS G12C , PDAC) (Fig. 4f,g and Extended Data Fig. 10e,f).As expected, downstream MEK1 mutations conferred resistance to both OFF state and ON state RAS inhibitors (Extended Data Fig. 10g).
Finally, we examined RMC-7977 treatment in a KRAS(G12C)-mutated PDX model derived from a NSCLC patient who had achieved stable disease on sotorasib but quickly relapsed.Genomic alterations in this tumour include amplification of the wild-type KRAS allele accompanied by increased levels of GTP-KRAS (M.Nokin et al., unpublished observations), which contributes to diminished response to sotorasib treatment at 50 mg kg −1 daily.RMC-7977 administered daily at 10 mg kg −1 resulted in significant anti-tumour activity, with 90% inhibition of tumour growth observed at day 17 of treatment, whereas sotorasib treatment induced only 47% tumour growth inhibition (Fig. 4h).In sum, these data indicate that both adaptive and acquired mechanisms of resistance to KRAS(G12C) inhibitors that lead to RAS pathway reactivation are susceptible to inhibition by RMC-7977.
RMC-7977 extends the tri-complex inhibitor strategy to noncovalently target the active state of wild-type and multiple oncogenic RAS variant proteins, with particular activity against the range of common codon 12 mutants, thus offering therapeutic potential for RAS(ON) multi-selective inhibitor across a spectrum of RAS-addicted cancers, including NSCLC, CRC and PDAC.Evidence of robust, durable anti-tumour activity at well-tolerated doses across various RAS mutant xenograft models provides preclinical validation for the direct targeting of active RAS variants as a desirable therapeutic strategy.Furthermore, we demonstrate that concurrent inhibition of multiple oncogenic RAS variants and wild-type RAS in the same tumour cell with a reversible broad-spectrum RAS MULTI inhibitor such as RMC-7977 can overcome some of the resistance mechanisms recognized to limit the efficacy and durability for inactive-state KRAS(G12C) inhibitors.The proximity of the RMC-7977 binding site to RAS mutational hotspots (residues G12, G13 and Q61) presents a unique opportunity to expand this approach further by designing additional mutant-selective tri-complex inhibitors.Moreover, RAS(ON) multi-selective inhibitors could also provide therapeutic benefit in combination with mutant-selective KRAS

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inhibitors to improve anti-tumour response by blocking adaptive pathway reactivation and preventing escape through emergence of secondary oncogenic RAS or RTK mutations.The investigational agent RMC-6236 is a first-in-class broad-spectrum RAS(ON) multi-selective protein inhibitor that is currently undergoing clinical evaluation (Clini-calTrials.govidentifier: NCT05379985).

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Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-024-07205-6.

Cell culture and reagents
Most cell lines were obtained from ATCC (listed in Supplementary Methods).Pa14C and Pa16C cells were a gift from A. Maitra, and the MEF cell lines were obtained from the NIH.AsPC-1 CYPA-knockout (KO), NCI-H441 CYPA-KO and eCT26 KRAS G12C/G12C ABCB1 −/− cells were generated by Synthego (eCT26 KRAS G12C/G12C ABCB1 −/− was engineered from the mouse CT26 KRAS G12D/G12D tumour cell line; P-glycoprotein (PGP) drug transporter was knocked out to eliminate any confounds due to potential interaction of the test article with PGP).All cells were grown in recommended medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintained at 37 °C in a humidified incubator at 5% CO 2 .The sanglifehrin A-competitive CYPA inhibitor 17 was synthesized at WuXi AppTec.Other tool inhibitors were acquired from Selleckchem or MedChemExpress.

Crystallography
Conditions, data collection, and refinement protocols are provided in the Supplementary Methods.

RAS-RAF and RAS-CYPA TR-FRET
Time-resolved fluorescence resonance energy transfer (TR-FRET) was used as previously described to assess disruption of the interactions between wild-type RAS or the mutant oncogenic RAS proteins and the RAS-binding domain of BRAF, and to assess the induction of interactions between the RAS proteins and CYPA 12 .

CYPA binding affinity
The binding affinity of compounds for CYPA (K d1 ) was assessed by SPR on a Biacore 8K instrument as previously described 12 .

RAS binding affinity
The binding affinity of compound-bound CYPA for the mutant oncogenic RAS proteins (K d2 ) mentioned was assessed by SPR on a Biacore 8 K instrument.AviTag-RAS [residues 1-169] was immobilized on a streptavidin sensor chip, and varying compound concentrations were flowed over the chip in assay buffer (10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 0.005% v/v surfactant P20, 2% v/v DMSO, 25 µM CYPA).The SPR sensorgrams were fit using either a steady state affinity model or a 1:1 binding (kinetic) model to assess the dissociation constant (K d ) for RAS binding.PSN1 and HUPT3, lower plateau = 0%).Pa16C MEK1 mutant cells were evaluated by live-cell counting using Calcein AM and a SpectraMax i3X multi-mode detection platform (Molecular Devices).Growth percentages were calculated by normalizing the treated cell counts to their respective untreated cell counts.

Cellular RAS-RAF and RAS-CYPA assays
U2OS cells or U2OS cells with PPIA gene knockout were seeded at 500,000 cells per well in a 6-well plate and incubated overnight.KRAS4B, or other small GTPases, containing the indicated mutations were cloned in pNLF-N or pHTN plasmids for expression with an N-terminal nanoluciferase or HaloTag fusion, respectively.Full-length CYPA was cloned into pHTN, the RBD of RAF1 (residues 51-149) was cloned into pHTC, full-length RALGDS was cloned into pHTC, PIK3CA was cloned into pNLF-N, and the catalytic domain of SOS1 (residues 558-1049) was cloned into pNLF-N.U2OS cells were transfected with KRAS and effector plasmids, and U2OS PPIA-KO cells were transfected with small GTPase and CYPA plasmids, both using Fugene HD reagent according to manufacturer protocols.The following day, the cells were collected by Trypsin and reseeded in a white tissue culture-treated 96-well plate in OptiMem phenol red-free medium (Gibco) containing 4% FBS and a 1:1,000 dilution of NanoBRET 618 HaloTag ligand (Promega).For endpoint concentration response curves, vivazine nanoluciferase substrate was added to 1× concentration in OptiMem phenol red-free medium with 4% FBS.Varying concentrations of inhibitor were added and incubated for 1 or 4 h before the nano-BRET signal was measured on a Perkin Elmer Envision plate reader.For kinetic assays, endurazine nanoluciferase substrate was used in place of vivazine, and the plate was placed in a Cytation5 multi-mode reader pre-equilibrated to 37 °C and 5% CO 2 .After 1 h of equilibration, RMC-7977 (50 nM) was added and the nano-BRET signal measured.

Generation of NCI-H358 expressing low and high CYPA
NCI-H358 cells were transduced by lentivirus encoding Cas9, a guide RNA targeting PPIA (which encodes CYPA), and the puromycin resistance gene at WarpDrive Bio.Following puromycin selection, Flag-CYPA was introduced under the control of a tet-inducible promoter by lentivirus.Clones with high and low expression levels of Flag-CYPA were isolated at Revolution Medicines.Flag-CYPA expression was induced by adding doxycycline (0.1 µg ml −1 ) for at least 24 h.

Generation of cell lines with acquired resistance to adagrasib
NCI-H358 cells resistant to adagrasib were generated by continuously culturing in growth medium containing 1 µM adagrasib for approximately 2 months.Resistant cells were subsequently maintained in culture medium containing 1 µM adagrasib, which was removed during assays.

Generation of Pa16C cells expressing MEK1 mutants
MEK1 mutants were generated using quick change mutagenesis in MEK1-GFP (Addgene plasmid #14746).MEK1 was PCR amplified with flanking NheI and AgeI sites and digested.Luciferase-PCW107-V5 (Addgene plasmid #64649) was also digested with NheI and AgeI, removing luciferase, and ligated with the MEK1 insert in front of the V5 tag.Lentivirus was made from the control construct (Luciferase-PCW107-V5) and each of the MEK1 constructs by co-transfection with psPax packaging plasmid into HEK293T cells using Fugene 6 Transfection Reagent (Promega).Viral supernatant was collected, combined with polybrene (8 µg ml −1 ), and used to transduce Pa16C (PDAC, KRAS(G12D)) cells in DMEM supplemented with 10% FBS.Cells were infected for 12 h and then selected using puromycin.

PRISM assay
RMC-7977 was screened in 931 PRISM DNA-barcoded cell lines established by the Broad Institute.In brief, 20-25 cell lines per pool were plated in 384 well plates and treated with RMC-7977 at 8 doses in threefold dilutions starting at 10 µM for 5 days.Cells were then lysed in TCL mRNA lysis buffer, and then PCR with reverse transcription was performed.Detection of the barcodes and univariate and multivariate analysis was then performed as previously described 43 .Data analysis is described in the Supplementary Methods.Up-to-date code for our analysis is at the github link: https://github.com/cmap/docker-ized_mts.

Western blot analysis
Antibodies and protocols are described in the Supplementary Methods.

Quantification of CYPA protein level in cell and tumour samples
Cells were seeded at 1 × 10 6 cells per well in triplicate in 6-well plates and incubated overnight.The following day, cells were collected by Trypsin, washed in PBS, pelleted by centrifugation, and snap frozen in a slurry of dry ice and ethanol.Tumour samples were collected and flash frozen (see Supplementary Methods).Samples were transferred to IQ Proteomics for analysis.The samples were lysed by bead beating in 8 M urea + 200 mM EPPS pH 8.0 + HALT protease inhibitor cocktail.Following bead beating, SDS was added to the lysate, 1% final (w/v).Following quantification by BCA assay, lysate corresponding to 16 µg of total protein was aliquoted for downstream processing.Samples were reduced and alkylated via DTT/Iodoacetamide, and protein was isolated via ethanol precipitation.Protein was digested in 100 mM EPPS pH 8.1, using LysC (overnight, room temperature) and Trypsin (6 h, 37 °C). 5 stable isotope labelled standard peptides spanning the CYPA protein sequence (sequences VSFELFADK, ALSTGEK, FEDENFILK, TEWLDGK and EGMNIVEAMER) were spiked into each sample at a ratio of 25 fmol µg −1 total protein digested.Endogenous (light) and internal standard (heavy) peptides were quantified via custom targeted assay on an Orbitrap Lumos instrument (Thermo).

Bioanalysis of cells and supernatant
Ten-million cells were exposed to RMC-7977 (10, 100 or 1,000 nM) in suspension at 1 × 10 6 cells ml −1 for 1 h at 37 °C.Cells were pelleted by centrifugation, and 1 ml of supernatant was reserved and frozen at −80 °C.Cell pellets were washed twice in cold PBS, and pre-weighed tubes containing the cell pellets were weighed prior to snap freezing in a slurry of dry ice and ethanol.Concentrations of RMC-7977 in cell pellets and supernatant were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods.Cell pellet samples were resuspended in cell medium (diluted as needed), then treated as supernatant.An aliquot of supernatant or resuspended cells (50 µL) was quenched with a 3× volume of acetonitrile containing the internal standard terfenadine (2.5 ng ml −1 ).Samples were vortexed, centrifuged, and analysed on a Sciex 6500+ triple quadrupole mass spectrometer equipped with a Shimadzu AD LC system.A Waters ACQUITY UPLC BEH C4 1.7 µm (2.1 × 50 mm) column was used with gradient elution for compound separation.RMC-7977 and internal standard were detected by positive electrospray ionization using multiple reaction monitoring (RMC-7977: m/z 865.273/833.500; terfenadine: m/z 471.939/436.300).The lower limit of quantification was 0.25 ng ml −1 , and the calibration range was 0.25 to 400 ng ml −1 .
The intracellular concentration of RMC-7977 was calculated using the mass of each cell pellet (mass of empty tube subtracted) and the known cell number, with the assumptions that the volume of a cell is ~2,000 µm 3 , that the density of a cell is approximately the density of water (thus, cell volume = cell mass); and that any compound in CYPA-KO cells in excess of the medium concentration is probably membrane-bound.The ratio of compound concentration in the cell pellet to compound in medium was determined for each concentration of RMC-7977 tested.

Animal studies
Xenograft studies were conducted at GenenDesign, Pharmaron, Wuxi AppTec, the laboratory of P. Lito, and the laboratory of C. Ambrogio.Animals were assigned to study groups using stratified randomization based upon their tumour volumes.All procedures related to animal handling, care and treatment were conducted in compliance with all applicable regulations and guidelines of the relevant Institutional Animal Care and Use Committee (IACUC).For the sotorasib-resistance xenograft study, all procedures and animal housing conformed to the regulatory standards and were approved by the Italian Health Minister (authorization no.1227/2020-PR); all experiments were performed in accordance with the guideline for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research Involving Animals, developed by the Council for International Organizations of Medical Sciences.Experimental details are supplied in the Supplementary Methods.

Mouse blood and tumour sample bioanalysis
Whole-blood and tumour concentrations of RMC-7977 were determined using LC-MS/MS methods performed at WuXi AppTec.Tumour tissue samples were homogenized with a 10× volume of methanol/15 mM PBS (1:2, v:v).Sample preparation and analysis on a Sciex 6500+ triple quadrupole mass spectrometer equipped with an ACQUITY UPLC system were performed as previously described 12 .RMC-7977 and internal standard verapamil were detected by positive electrospray ionization using multiple reaction monitoring (RMC-7977: m/z 865.4/706.4;verapamil: m/z 455.2/164.9).

In vivo pharmacodynamic analysis by DUSP6 qPCR
RNA extraction and analysis of DUSP6 levels by in tumour tissue were performed as previously described 12 .

OVA peptide vaccination
Experimental details are described in Supplementary Methods.

Immune cell response in vivo
Experimental details are described in Supplementary Methods.or HRAS (f) gene effect (see Methods).g, Mean KRAS Chronos score for each KRAS genotype is shown, with the mean effect score across all cell lines subtracted.P-values were calculated by a two-sided Wilcoxon rank sums test comparing the distribution of genotype effect to the distribution of effect scores outside of that genotype (Bonferonni-corrected p-value of 0.05/31 = 0.0016 indicated by a gray horizontal line, 31 KRAS genotypes tested, point size is proportional to sample size of each genotype, see methods). )

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g.means) or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.
Crystallography: All data were processed with XDS, and initial structures were determined via Phaser using previously solved KRAS and CYPA as molecular replacement search models.Ligand restraints were generated using AceDRG5.The final structures were determined through iterative rounds of model building using Coot and refinement using REFMAC5 from the CCP4 suite and phenix.refine.
Western blots: Image Studio Lite v.5.2.5) and Image Lab (v.6.1.0build 7) were used for analysis Inhibitor response modeling, curve fitting, generation of graphs: Prism 9 (GraphPad) was used to plot data, estimate EC50 or IC50, and display data in graphical form.
Flow cytometry: Data was acquired using SpectroFlo (version 3.1.2)and analyzed with FlowJo (version 10.10) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.

Fig. 2 |
Fig. 2 | RAS inhibition is CYPA-dependent and active against multiple RAS variants.a,b, Formation of KRAS-CYPA complexes and disruption of the KRAS(G12V)-CRAF interaction in U2OS cells as a time course after 50 nM RMC-7977 treatment, expressed as % of the maximum (max) signal (a), and correlation between potency of RAS-RAF inhibition and formation of the tri-complex for multiple KRAS variants (R 2 = 0.7) (b).Data points are single nano-BRET measurements representative of three independent experiments.c, Proliferation (measured by CellTiter-Glo (CTG) assay) of NCI-H358 cells with doxycycline (Dox)-inducible expression of low or high CYPA levels treated with RMC-7977 for 120 h.Data points show biological duplicates normalized to vehicle control.Representative data from one of three independent experiments.d, Liquid chromatography-mass spectrometry measurements of the ratio of RMC-7977 concentration in CYPA-high and CYPA-low NCI-H358 volume (% change from baseline)

Fig. 3 |
Fig. 3 | RMC-7977 is broadly active in RAS-addicted cancer models.a, Relationship between the area under the curve (AUC) difference (see Supplementary Methods) and negative log-transformed P value (two-sided Wilcoxon test) between cell lines by genotype.Points represent mutated genes.Negative AUC indicates sensitivity; positive AUC indicates resistance.b, RMC-7977 EC 50 according to KRAS genotype.Each dot represents a cell line.The centre line is the median, box limits represent first and third quartiles and whiskers depict the range.The number of cell lines in each group is indicated in parentheses.VUS, variants of unknown significance.c, Blood and tumour concentrations of RMC-7977 (green) and DUSP6 mRNA (blue) for NCI-H441 xenograft tumours following one oral dose of 10 mg kg −1 RMC-7977.Data are mean ± s.e.m. of three biological replicates.d, Mice bearing NCI-H441 CDX tumours treated with 10 mg kg −1 RMC-7977 orally once daily for 28 days.***Adjusted P value = 0.0002; two-way ANOVA (n = 8 mice per group) with multiple comparison Dunnett's test.The dashed line shows the initial average tumour volume.Data are mean ± s.e.m. for eight mice per group.e, KRAS(G12X)xenograft models treated with RMC-7977 (10 mg kg −1 by oral administration) for 4-6 weeks.Data are mean ± s.e.m. of 3-18 mice per group.One data point for LUAD G12C is beyond the axis range.Shaded boxes in the table indicate gene variants.f, Kaplan-Meier analysis of time to tumour size doubling (n = 90 mice per group) of KRAS G12X mutant models treated with 10 mg kg −1 RMC-7977 orally once daily.g, CDX models treated with vehicle control, SHP2 inhibitor (20 mg kg −1 RMC-4550 orally every 2 days), MEK inhibitor (2.5 mg kg −1 cobimetinib orally once daily), combined SHP2 and MEK inhibitors (20 mg kg −1 RMC-4550 orally every 2 days and 2.5 mg kg −1 cobimetinib orally once daily), or 10 mg kg −1 RMC-7977 orally once daily.NCI-H441 (KRAS G12V , NSCLC) and HPAC (KRAS G12D , PDAC) models were treated for 21 days.SW620 (KRAS G12V , CRC) was treated for 28 days.Data are mean ± s.e.m.; n = 8 mice per group for control and RMC-7977, and n = 10 mice per group for RMC-4550, cobimetinib, and RMC-4550 + cobimetinib.

Fig. 4 |
Fig. 4 | RMC-7977 can overcome resistance to mutant-selective KRAS inhibition.a, Western blots showing the time course of RAS signalling in KRAS G12D PDAC cell lines treated with RMC-7977, MRTX1133 or DMSO control.Total ERK and vinculin were used as loading controls.Data are representative of two similar experiments.b,c, Parental NCI-H358 cells (KRAS G12C , NSCLC) (b) and adagrasib-resistant NCI-H358 cells with a secondary NRAS Q61K mutation (c) were treated with adagrasib or RMC-7977 for 5 days and proliferation was measured by CTG assay.d,e, NCI-H358 (KRAS G12C , NSCLC) cells expressing exogenous RTK DNA constructs as indicated (GFP control, wild-type EGFR, EGFR(A289V), HER2, FGFR2 or RET(M918T)) were treated with adagrasib (d) or RMC-7977 (e) for 120 h, and proliferation was measured by CTG assay.f,g, MIA PaCa-2 (KRAS G12C , PDAC) cells expressing exogenous RTK fusion DNA constructs as indicated (GFP control, EML4-ALK, CCDC6-RET or FGFR3-TACC3) were treated with adagrasib (f) or RMC-7977 (g) for 120 h, and proliferation was measured by CTG assay.d-g, Biological duplicates normalized to vehicle control are shown from one of 2-5 independent experiments.h, Patient-derived xenograft model established from a patient with KRAS G12C NSCLC who developed resistance after sotorasib.Mice were treated with vehicle (n = 7), sotorasib (50 mg kg −1 orally once daily; n = 7), or RMC-7977 (10 mg kg −1 orally once daily; n = 10).Tumour volumes were assessed for 17 days after treatment started.***Adjusted P value = 0.0001 for RMC-7977 versus control group; repeated measures two-way ANOVA adjusted based on multiple comparison via Dunnett's test on the final tumour measurement.Data are mean ± s.e.m. n refers to the number of mice in each group.

. 3 |. 4 |. 5 |. 6 |
RAS effector inhibition and tri-complex selectivity.a, Recombinant KRAS and the RALGDS RID treated with the indicated concentrations of RMC-7977 and CYPA (KRAS G12V IC 50 = 8 nM, WT KRAS IC 50 = 14 nM, points represent mean ±s.d. of 4 replicates) b,c,d, Cellular nano-BRET assays for multiple RAS-binding proteins, including full length RALGDS (b), full length PI3Kα (c) binding to KRAS G12C, G12D, and G12V, and the catalytic domain of SOS1 (d) binding to KRAS WT.U2OS cells were treated with RMC-7977 for 1 h.Points represent mean ±s.d. of 6 replicates e, Sequence identity analysis of related GTP-ase proteins using KRAS residues positioned within 4 angstroms of RMC-7977 in the tri-complex co-structures as a reference sequence.f, Cellular miliBRET signal between CYPA and 5 different small GTPase proteins with moderate to high homology to the KRAS tri-complex interface (RIT1, MRAS, RRAS, RRAS2, and Rheb) treated with RMC-7977.MRAS, RRAS and RRAS2 oncogenic mutants used to induce the active, GTP-bound state.Points represent mean ±s.d. of 6 biological replicates.Cellular concentration of CYPA determines the cellular concentration binary complex.a, Ratio of RMC-7977 concentration in cells to concentration in media following exposure of parental or PPIA KO AsPC-1 cells to indicated concentrations of RMC-7977 for 1 h as determined by LC/ MS bioanalysis.Bars represent mean of the 3 biological replicates shown from one experiment.b, RMC-7977 concentration response for biochemical (points are the mean of biological duplicates from one of 6 independent experiments) and cellular (3 independent experiments) nano-BRET KRAS G12V -RAF disruption.Data shown are representative of independent replicates.c, same data as b, with calculated concentration of active binary complexes.Correction is based on equation 1: assumes the extracellular volume is much greater than the intracellular volume and that the concentration of unbound RMC-7977 ([RMC-7977] unbound ) equilibrates between intracellular and extracellular space.A value of 5 µM was used for the cellular CYPA concentration ([CYPA] cell ).No adjustment was applied to the biochemical data because under experimental conditions >99% of RMC-7977 is bound to CYPA.d-i, pERK levels (MSD) (d,f,h) and cell proliferation (CTG) (e,g,i) in AsPC-1 and NCI-H441 cells treated with RMC-7977 or trametinib for 4 h.RMC-7977 co-treatment with a CYPA inhibitor12 (d,e) or PPIA KO (f,g) rescued pERK and proliferation in RMC-7977 treated cells, but did not affect response to trametinib (h,i).Representative data from one of 2 (NCI-H441) or 3 (AsPC-1) independent experiments are shown.CYPA is required for cellular activity.a, Cellular CYPA protein levels determined by parallel reaction monitoring (PRM) mass spectrometry in NCI-H358 cells harboring doxycycline-inducible expression of low or high CYPA in the absence or presence of doxycycline (0.1 µg/ml) for 120 h.Bars indicate the mean of 3 biological replicates per group from one experiment.b, pERK levels (MSD) of CYPA high and CYPA low cells treated with RMC-7977 in the absence or presence of doxycycline (0.1 µg/ml) for 120 h.Datapoints show biological duplicates normalized to vehicle control.Representative data shown from one of 3 independent experiments.c, Cellular CYPA protein concentration in a panel of cell lines determined by PRM.Data 2 stable isotope labeled peptide standards (SIS) was averaged for each replicate.Intracellular µM concentration estimated assuming cell volume of 2 pl.Bars indicate the mean of biological duplicates.d, CYPA protein expression in cells (green bars) and corresponding xenograft tumours (blue bars) determined by PRM. 5 SIS peptides were used and data averaged for each biological replicate.Bars indicate the mean of biological duplicates for HPAC cells and triplicates for all others.e, PRISM screen, RMC-7977 sensitivity (AUC) by PPIA gene expression (RPKM).Each dot represents a cell line (n = 606).Two-sided Pearson correlation = −0.10(p = 0.011).Inhibition of RAS signal transduction pathways.a, Western blots of isogenic RAS-less MEF cell lines harboring an exogenous, wild-type (WT) or mutant KRAS, or BRAF V600E transgene, treated with indicated concentrations of trametinib or DMSO control for 24 h.Data shown are representative of 3 independent experiments.b,c, Western blots of KRAS mutated cell lines treated with RMC-7977 at indicated concentrations for 4 h (b), or treated with RMC-7977 (100 nM) or DMSO control for indicated time points.c, Data shown are representative of 2 independent experiments.

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Corresponding author(s):Mallika Singh, David Wildes, Jacqueline A.M. Smith Last updated by author(s):Jan 22, 2024   Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish.This form provides structure for consistency and transparency in reporting.For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist.

Extended Data Table 1 | Compound potencies and properties
Potencies for compounds 1-4 and RMC-7977 in biophysical (K D 1 and K D 2 measured by SPR), biochemical (KRAS:BRAF RBD disruption measured by time-resolved fluorescence binding assay), cellular, and in vivo assays (RAS:CRAF RBD Disruption and RAS:CYPA Binding measured by nano-BRET, pERK measured by MSD, proliferation measured by CTG, Oral bioavailability measured by LC-MS/MS analysis, 0-24-hour AUC, of total blood exposure from a single oral dose compared to intravenous exposure).n=number of independent experiments performed.

Table 3 | RMC-7977 NRAS and HRAS potencies
Potencies for RMC-7977 in biophysical (steady state K D 2 measured by SPR), biochemical (KRAS:BRAF RBD disruption measured by time-resolved fluorescence binding assay).n=number of independent experiments performed.