RAS family: KRAS(G12C); c-Raf; H-Ras; NRAS rG4

RMC-7977 is 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).[1]

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RMC-7977 suppressed cell proliferation in AML cell lines driven by FLT3-ITD (Molm-14, MV4-11), KITN822K (Kasumi-1, SKNO-1) and RAS mutations ( NRASQ61L -OCIAML-3, HL-60, KRASG13D - NOMO-1) with IC 50 values between 5 and 33 nM and repressed phosphorylation of downstream effectors of the MAPK pathway MEK, ERK and RSK. We also assessed the activity of RMC-7977 in resistant Molm-14 cells that developed secondary NRASG12C or NRASQ61K mutations after long-term exposure to FLT3i. At concentrations as low as 5 nM, RMC-7977 restored sensitivity to gilteritinib in both NRAS-mutant resistant cell lines. Using a caspase3/7 assay, we observed that RMC-7977 induced apoptosis, but to different extents across AML cell lines. We have previously demonstrated that effective MAPK signaling inhibition increases apoptotic dependency on BCL2. Consequently, the addition of venetoclax to RMC-7977 significantly enhanced caspase activation in cell lines with FLT3, KIT and NRAS mutations. In a cell viability assay, RMC-7977 and venetoclax showed high synergistic activity as assessed by Bliss independence model.[2]

Given that intrapatient tumor heterogeneity is associated with clinical resistance to targeted therapies, researchers investigated the in vitro activity of RMC-7977 in models that recapitulate patterns of clonal outgrowth observed in patients treated with FLT3i. We mixed fluorescently-tagged cell lines with FLT3-ITD (Molm-14), FLT3-ITD and NRAS co-mutations (Molm-14 NRASQ61K) and NRAS-only mutations (OCIAML-3), treated the mixtures with RMC-7977 alone and in combination (with gilteritinib or venetoclax) for 96 hours, then assessed cell viability via flow cytometry. Gilteritinib and the gilteritinib/venetoclax combination selected for survival of cells harboring NRAS mutations, but RMC-7977 inhibited outgrowth of all cell populations. The combination of RMC-7977 and gilteritinib had superior activity in mixtures containing FLT3 and FLT3- NRAS co-mutations, but no additional benefit over RMC-7977 monotherapy in cells harboring NRAS mutations alone. Strikingly, the combination of RMC-7977 and venetoclax potently suppressed cell viability equally in all cell line models and was significantly superior to RMC-7977 alone. In vivo studies investigating the tolerability and activity of RMC-7977 and RMC-7977 combinations in RAS mutant/FLT3i-resistant patient-derived xenograft models are ongoing and will be presented.[2]

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).[1]

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In this study, researchers assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance [3].

RAS–RAF and RAS–CYPA TR-FRET[1]
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 CYPA12.

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CYPA binding affinity[1]
The binding affinity of compounds for CYPA (Kd1) was assessed by SPR on a Biacore 8K instrument as previously described12.

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RAS binding affinity[1]
The binding affinity of compound-bound CYPA for the mutant oncogenic RAS proteins (Kd2) 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 (Kd) for RAS binding.

Cellular RAS–RAF and RAS–CYPA assays[1]
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. 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% CO2. After 1 h of equilibration, RMC-7977 (50 nM) was added and the nano-BRET signal measured.

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PRISM assay[1]
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 described43. Data analysis is described in the Supplementary Methods. Up-to-date code for our analysis is at the github link: https://github.com/cmap/dockerized_mts. Cell panel[1]
A panel of 183 cancer cell lines harbouring mutant and wild-type RAS was screened for response to RMC-7977 by cell proliferation and viability inhibition at Crown Bioscience. The panel consisted of cell lines with any substitution at position 12 of KRAS, NRAS or HRAS (KRAS(G12X), NRAS(G12X), HRAS(G12X)); substitutions in KRAS, NRAS or HRAS at any position other than 12, 13 and 61 (KRAS(other/VUS), NRAS(other/VUS), HRAS(other/VUS)); other oncogenic mutations in the RAS pathway (ABL1, ALK, ARAF, BRAF, CBL, EGFR, ERBB2, ERBB3, ERBB4, ERRFI1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, HRAS, IGF1R, JAK2, KIT, MAP2K1, MAP2K2, MAPK1, MET, NF1, NRAS, NTRK1, NTRK2, NTRK3, PDGFRA, PTPN11, RAC1, RAF1, RASA1, RET, RIT1, ROS1 and SOS1); and no oncogenic mutations in the RAS pathway. Cells were cultured in methylcellulose and treated in triplicate with serial dilutions of RMC-7977 or DMSO. Cells were incubated for 120 h, and cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay (CTG) according to the manufacturer’s instructions. Data were plotted as a function of log [inhibitor (M)] and a four-parameter sigmoidal concentration response model was fitted to the data to estimate the inhibitor EC50 using Genedata Screener.

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Cell proliferation analysis[1]
Cells were seeded in 384- or 96-well tissue culture-treated plates in 2D and incubated overnight. Alternatively, cells were seeded in round-bottom ultra-low attachment 96-well plates, centrifuged at 1,000 rpm for 10 min to pellet the cells, and incubated overnight or up to 72 h to allow for 3D spheroid formation. Cells were exposed to serial dilutions of compound or DMSO control (0.1% v/v) for 120 h. Cell viability was determined by CellTiter-Glo 2.0 reagent (2D CTG) (Promega, G9243) or 3D CellTiter-Glo reagent (3D CTG) according to the manufacturer’s protocols. Luminescence was detected using a SpectraMax M5 Plate Reader of Perkin Elmer Enspire. Luminescence signal was normalized to vehicle-treated wells (normalized signal (%) = (luminescence (treated)/mean luminescence (vehicle)) × 100%). For PSN1 and HUPT3, raw signal was normalized to vehicle control and a low-signal control compound ((sample signal – average low control signal)/(average vehicle signal – average low control signal) × 100%). For NCI-H441 and AsPC-1 cells treated with the combination of RMC-7977 and the sanglifehrin A competitive CYPA inhibitor (3 mM), luminescence signal was normalized to that of the CYPA inhibitor treatment-only control (normalized signal (%) = (luminescence (treated)/mean luminescence (CYPA inhibitor only) × 100%)).

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Bioanalysis of cells and supernatant[1]
Ten-million cells were exposed to RMC-7977 (10, 100 or 1,000 nM) in suspension at 1 × 106 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 µm3, 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.

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Mouse cell viability assays[3]
PDAC mouse cell lines with KrasG12C or KrasG12D mutations (treatment-naive or derived from RMC-7977-treated endpoint KPC tumours) were seeded at 2 × 103 in a 96-well plate. Cells were treated 24 h later with DMSO or serial dilutions of RMC-7977, ERK inhibitor (SCH772984) or MEK inhibitor (trametinib). Cell viability was evaluated 72 h later by measuring ATP levels using the CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer’s instructions. Alternatively (in experiments comparing naive and resistant cell lines), live cells were fluorescently labelled using Calcein AM (20-min incubation at 500 nMr) and counted using the SpectraMax i3X multimode detection platform. Technical triplicates were run for each biological replicate and a total of 3–4 biological replicates was done for each cell line. Growth percentage was calculated by normalizing drug-treated values to DMSO control, which was set to 100%. Four-parameter drug response curves were generated from biological replicates in GraphPad Prism. Mean ± s.d. was plotted for each tested dilution.

For synergy evaluation testing RMC-7977 treatment-naive and resistant cell lines, a similar protocol was used with following change: 24 h post cell line seeding, RMC-7977, IAG933 (Nantong Hi-future Biotechnology, 2714434-21-4), or combined treatment were added to the cells using the D300e digital dispenser. Mean synergy value for each cell line was calculated using Excess over Bliss method, using SynergyFinder package in R Studio.

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Human cell line proliferation assay[3]
19 PDAC cell lines were tested for sensitivity to RMC-7977 as part of a panel of human cancer cell lines of various histotypes screened at Crown Bioscience. These PDAC cell lines harboured KRASG12D, KRASG12V, KRASG12C, KRASQ61H and BRAFV487_P492delinsA mutations. To measure inhibition of cell proliferation, cells were cultured in methylcellulose and treated in triplicates with serial dilutions of RMC-7977 (top concentration of 1 µM) or DMSO dispensed by a Tecan D300e digital dispenser. Cells were incubated for 120 h prior to measurement of ATP levels using CellTiter-Glo. Technical triplicates were run for each biological replicate and a total of 3–4 biological replicates were done for each cell line. Growth percentage was calculated by normalizing drug-treated values to DMSO control, which was set to 100%. Normalized CTG assay readouts were plotted as a function of log molar inhibitor concentration and a four-parameter sigmoidal concentration–response model was fitted to the data. Mean ± s.d. was plotted for each tested dilution.

PDAC cell lines harbouring wild-type KRAS or KRASQ61H were plated at 500–4,000 cells per well in clear, flat-bottomed 96-well plates and grown for 24 h prior to adding indicated concentration of RMC-7977 or DMSO using the D300e digital dispenser. Following treatment, cells were incubated for additional 3–5 days after which live cells were fluorescently labelled using Calcein AM (20-min incubation at 500 nM) and counted using the SpectraMax i3X multimode detection platform. Experiments were day 0 normalized using an independent culture plate. Growth percentage was calculated by normalizing drug-treated values to DMSO control, which was set to 100%. Four-parameter sigmoidal concentration–response models were fitted to the data from at least three biological replicates. Mean ± s.d. was plotted for each tested dilution.

For synergy evaluation of combined RMC-7977 and IAG933, a similar protocol was used with following change: 24 h post cell line seeding, RMC-7977, IAG933 (2714434-21-4), or the combinations were added to the cells using the D300e digital dispenser (Tecan). Each cell line was considered as a separate biological replicate (n = 8). Mean synergy value for each cell line was calculated using the excess over bliss method, using SynergyFinder package in R(Studio). Western blot analysis[3]
Cells were seeded at 7.5 × 103 to 4 × 106 cells per well in 6-well plates or 100-mm dishes in growth medium. After overnight incubation, indicated compound (RMC-7977, IAG933 or DMSO (0.1% v/v)) were added and incubated for the indicated timepoints. Cells were washed twice with ice-cold PBS and lysed with NP-40 lysis buffer (Thermo Fisher, J60766), MSD Tris Lysis Buffer (MSD, R60TX-2), RIPA buffer (50 mM TRIS-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) or a lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA. All lysis buffers were supplemented with protease and phosphatase inhibitors. Lysates were scraped and collected before centrifugation at 21,000g for 10 min at 4 °C. The protein-containing supernatants were quantified by BCA assay and equal quantities of protein were denatured with LDS and reducing agent at 95 °C. Samples were resolved on 12% or 4–12% Bis-Tris polyacrylamide gels, then transferred to a nitrocellulose or PVDF membrane using the iBlot 2.0 system or wet transfer. Membranes were blocked in Intercept TBS buffer (Li-Cor, 927-60001) or 3-5% milk before probing with primary antibodies overnight at 4 °C. Secondary antibodies were added as appropriate, and the membranes were imaged on a Li-Cor Odyssey imager. Alternatively, membranes were incubated with HRP-linked secondary antibodies and developed with Clarity or ClarityMax chemiluminescent substrates using a ChemiDoc XRS+ or ChemiDoc MP imager.

RMC-7977 formulation[3]
For in vitro studies RMC-7977 was re-suspended in DMSO (Fisher Bioreagents, BP231-100) and used at 10 mM stock concentration. For use in the in vivo studies RMC-7977 was prepared using the formulation made of 10/20/10/60 (%v/v/v/v) DMSO/PEG 400/Solutol HS15/water. The same vehicle formulation was used for all control groups.

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In vivo xenograft studies[3]
RMC-7977 treatment Tumour-bearing mice were randomized and assigned into groups (n = 3–10 per group). Vehicle or RMC-7977 was administered via oral gavage daily at 10 mg kg−1 and mice were treated for 21–28 days. Studies were terminated early if tumour burden reached humane endpoint. Body weights were collected twice a week during the study. Means ± s.e.m were plotted in the waterfall plots. For the single-dose pharmacokinetic–pharmacodynamic study, mice were randomized and assigned into groups (n = 3–6 per dose and timepoint). A single dose of RMC-7977 was administered orally at 10 mg kg−1, 25 mg kg−1 or 50 mg kg−1. Tissues (including tumour, colon and skin) were collected at indicated timepoints and either fixed in 10% formalin, embedded in Optimal Cutting Temperature (OCT; Sakura, 4583) solution or snap-frozen in liquid nitrogen for further analysis. Whole blood was transferred into K2EDTA Microtainer tubes (BD, 365974), incubated for 5 min and snap-frozen in liquid nitrogen.

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In vivo allograft studies[3]
Mouse studies: All mouse allograft studies and procedures related to animal handling, care and treatment were conducted in compliance with all applicable regulations and guidelines of the Institutional Animal Care and Use Committee (IACUC). Female C57BL/6J (strain 000664) mice aged 6–8 weeks from the Jackson Laboratory were used for these studies. Generation of allograft models In order to generate subcutaneous allograft tumours, each mouse was inoculated in the right flank with 3 × 105 of KPCY 6499c4 tumour cells in 0.1 ml of Matrigel:PBS (1:1). Treatments were started when the average tumour size reached 140 mm3. Tumour size was measured at two dimensions using a digital calliper, and the tumour volume in mm3 was calculated using the formula volume = (width2 × length)/2. Mice on studies were weighed and tumours were measured 2 times a week. To generate orthotopic allograft tumours, 5 × 104 KPCY 6499c4 tumour cells in 20 µl PBS/Matrigel mixtures (1:1) were implanted directly into the mouse pancreas through a laparoscopic incision. Treatments were started when the average tumour size reached ~50 mm3. Body weights were measured and tumour growth was monitored by ultrasound twice weekly.
RMC-7977 treatment: Tumour-bearing mice were randomized, assigned into groups (n = 9–10 per group), and treated daily via oral gavage with vehicle or RMC-7977 (10 mg kg−1). For subcutaneous KPCY study, survival endpoint was defined as: tumour volume reaching 2000 mm3 or mice showing any clinical signs, including severe ulceration. For orthotopic KPCY study, survival endpoint was defined as (1) mice showing any clinical signs including hunching or fluid in the abdomen, or (2) tumour dimensions exceeding the imaging frame of the ultrasound. Body weights were measured twice a week during the study. Tissue was collected either at 4 h or 24 h after last dose and preserved as previously described (see ‘In vivo xenograft studies’).

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In vivo GEMM studies[3]
Pharmacokinetic–pharmacodynamic study in KPF/FC[3]
Tumour formation in KPF/FC mice was monitored by bi-weekly palpations until the detection of a mass, which was then confirmed by ultrasound. Tumour-bearing mice were randomized and assigned into groups (n = 3 per dose and timepoint). Single dose of vehicle or RMC-7977 was administered orally at 10 mg kg−1, 25 mg kg−1 or 50 mg kg−1. Whole blood and tissue (tumours and colons) were collected at indicated timepoints and preserved as previously described (see ‘In vivo xenograft studies’).

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Pharmacodynamic study in KPC mice [3]
Tumour formation in KPC mice was monitored by bi-weekly palpation. Upon detection of a 4–7 mm diameter tumour by ultrasound, KPC mice were randomized and treated with vehicle (n = 6) or RMC-7977 (50 mg kg−1; n = 11). Treatments were performed every other day via oral gavage for 1 week. Mouse health status and weight were checked daily and ultrasounds (Vevo 3100) were performed every third day to monitor tumour growth. Following two consecutive ultrasounds, RMC-7977-treated mice were euthanized either 4 (n = 7) or 24 h (n = 4) after last dose and vehicle-treated mice were euthanized between 4–24 h post last dose. Tissue was collected and preserved as previously described (see ‘In vivo xenograft studies’). Additional group of KPC mice was also treated with a single dose of RMC-7977 (n = 10) or vehicle (n = 3) and tissues were collected at 4 or 24 h post-dose as previously described.

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Pharmacodynamic study in KPCY mice [3]
KPCY mice were enroled upon detection of a 15–100 mm3 tumour measured via ultrasound. Mice were randomized into groups and treated with vehicle (n = 6) or RMC-7977 (25 mg kg−1; n = 8). Treatments were performed every day via oral gavage for 15 days and ultrasounds were performed on day 8 and 15. Mice were euthanized after last dose and tissue was collected and preserved as previously described (see ‘In vivo xenograft studies’).

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Survival study in KPC mice [3]
For survival study, KPC mice with 4–7 mm diameter tumours (as measured by ultrasound) were enroled and treated every other day with vehicle (n = 9) or RMC-7977 (50 mg kg−1; n = 13). Mouse health status and weight were checked daily and ultrasounds were performed every third day to monitor tumour growth. The survival endpoint was determined by overall health criteria scoring, where endpoint is determined by a score of 5 or greater based on the following criteria: moribund, immediate euthanasia; abdominal distention due to haemorrhagic ascites, 5 pts; mild difficulty beathing, 5 points; hypothermia, 5 points; abdominal distention due to chylous ascites, 3 points; loss of over 20% enrolment body weight, 3 points; failure of grasp test, 3 points; jaundice or pallor, 3 points; weak grasp test, 2 points; failure to interact with other mice, 1 point; hunched, 1 point; pilorection/failure to groom, 1 point.

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Mouse blood and tumour sample bioanalysis[1]
Whole-blood and tumour concentrations of RMC-7977 were determined using LC–MS/MS methods. 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 described12. 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).

[1]. Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy. Research Square; 2023.

[2]. RAS MULTI (ON) inhibitor RMC-7977 targets oncogenic RAS mutations and overcomes RAS/MAPK-mediated resistance to FLT3 inhibitors in AML models. Blood, 2023, 142: 2793.

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).[1]

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FLT3 inhibitors (FLT3i) such as gilteritinib are clinically active in AML, but their use is limited by resistance due to emergence of clones with RAS/MAPK mutations, both alone and in combination with FLT3 mutations, or with other drivers such as KIT mutations. RAS mutations are also commonly associated with relapse on IDH1/2 inhibitors and the BCL2 inhibitor venetoclax. Importantly, multiple heterogeneous resistant clones are identified at the time of relapse. In addition, 5-10% of all de novo patients harbor oncogenic RAS mutations. Patients with RAS mutations or other mutations that activate RAS/MAPK signaling do not benefit from clinically approved targeted therapies. Targeting oncogenic RAS has been historically challenging and inhibition of downstream effectors of the MAPK pathway, such as MEK, demonstrated modest activity and high toxicity in clinical trials. Effective inhibition of the RAS/MAPK pathway is therefore a critical unmet need in AML. RMC-7977 is a potent, oral small molecule inhibitor of both wild-type and mutant GTP-bound RAS oncoproteins (RAS MULTI) and is a preclinical tool compound representative of the clinical candidate RMC-6236, currently in clinical evaluation (NCT05379985). RMC-7977 non-covalently binds to the intracellular chaperone cyclophilin A, generating a neomorphic interface with high affinity for all isoforms of RAS. The resulting tri-complexes sterically block RAS-effector interactions required for propagating oncogenic signals. We report in vitro data supporting preclinical utility of RAS MULTI(ON) inhibition in AML models harboring RAS mutations, including those with resistance to FLT3i due to hyperactive RAS signaling.Given that intrapatient tumor heterogeneity is associated with clinical resistance to targeted therapies, we investigated the in vitro activity of RMC-7977 in models that recapitulate patterns of clonal outgrowth observed in patients treated with FLT3i. We mixed fluorescently-tagged cell lines with FLT3-ITD (Molm-14), FLT3-ITD and NRAS co-mutations (Molm-14 NRASQ61K) and NRAS-only mutations (OCIAML-3), treated the mixtures with RMC-7977 alone and in combination (with gilteritinib or venetoclax) for 96 hours, then assessed cell viability via flow cytometry. Gilteritinib and the gilteritinib/venetoclax combination selected for survival of cells harboring NRAS mutations, but RMC-7977 inhibited outgrowth of all cell populations. The combination of RMC-7977 and gilteritinib had superior activity in mixtures containing FLT3 and FLT3- NRAS co-mutations, but no additional benefit over RMC-7977 monotherapy in cells harboring NRAS mutations alone. Strikingly, the combination of RMC-7977 and venetoclax potently suppressed cell viability equally in all cell line models and was significantly superior to RMC-7977 alone. In vivo studies investigating the tolerability and activity of RMC-7977 and RMC-7977 combinations in RAS mutant/FLT3i-resistant patient-derived xenograft models are ongoing and will be presented. Collectively, our data provide preclinical evidence that combination therapies leveraging RAS MULTI(ON) inhibition are effective in suppressing RAS-mutant AML clones, a common mechanism of resistance to currently approved targeted therapies in AML and a current area of high unmet clinical need.[2]

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Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS.[3]

C47H60N8O6S

865.094309806824

864.44

C, 65.25; H, 6.99; N, 12.95; O, 11.10; S, 3.71

2765082-12-8

164726623

White to light yellow solid powder

4.7

2

12

8

62

1620

5

CCN1C2=C3C=C(C=C2)C4=CSC(=N4)C[C@@H](C(=O)N5CCC[C@H](N5)C(=O)OCC(CC3=C1C6=C(N=CC(=C6)N7CCN(CC7)C8CC8)[C@H](C)OC)(C)C)NC(=O)C9[C@H]1[C@@H]9COC1

NBLZKEHVVJSAAY-OFTZCYAMSA-N

InChI=1S/C47H60N8O6S/c1-6-54-39-12-9-28-18-31(39)33(43(54)32-19-30(22-48-42(32)27(2)59-5)53-16-14-52(15-17-53)29-10-11-29)21-47(3,4)26-61-46(58)36-8-7-13-55(51-36)45(57)37(20-40-49-38(28)25-62-40)50-44(56)41-34-23-60-24-35(34)41/h9,12,18-19,22,25,27,29,34-37,41,51H,6-8,10-11,13-17,20-21,23-24,26H2,1-5H3,(H,50,56)/t27-,34-,35+,36-,37+,41+/m0/s1

(1R,5S,6r)-N-((63S,4R,Z)-12-(5-(4-cyclopropylpiperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-11-ethyl-10,10-dimethyl-5,7-dioxo-61,62,63,64,65,66-hexahydro-11H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-4-yl)-3-oxabicyclo[3.1.0]hexane-6-carboxamide

2765082-12-8; SCHEMBL25774785; RMC-7977; RMC 7977; RMC7977; EX-A7974;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)