Journal: Cancer Discovery

Article Title: Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers

doi: 10.1158/2159-8290.CD-24-0027

Figure Lengend Snippet: RMC-6236 is a potent noncovalent inhibitor of the GTP-bound state of multiple RAS variants in vitro . A, Chemical structure of RMC-6236. B, Biochemical potency of RMC-6236 for wild-type KRAS, NRAS, HRAS, and several oncogenic RAS variants. EC 50 values shown for inhibition of RAS-RAF binding using recombinant proteins in vitro . Error bars indicate ± 95% CI. C, Immunoblot protein Western analyses of KRAS pathway targets in HPAC ( KRAS G12D/WT , PDAC) and Capan-2 ( KRAS G12V/WT , PDAC) cancer cells treated with RMC-6236 at the indicated concentrations and time points. D, RMC-6236 potency measured in the PRISM panel of cancer cell lines. Left, AUC difference between cell lines with and without a given gene mutation (x-axis) and the significance of the difference (y-axis). Points represent mutated genes. A negative AUC indicates increased sensitivity to RMC-6236 and positive AUC indicates resistance. Horizontal dashed line represents the P -value cutoff of 5 × 10 −8 . Vertical lines represent the absolute effect cutoff of 0.1. Right: AUC for KRAS mutant [glycine 12 depicted as KRAS G12X (115 lines); all other KRAS mutations labeled KRAS Other (42 lines)], NRAS mutant [glutamine 61 depicted as NRAS Q61X (34 lines); all other NRAS mutations labeled as NRAS Other (20 lines)], HRAS mutant, NF1 mutant, EGFR mutant, PTPN11 mutant, and BRAF V600E mutant cell lines are shown. Comparison of indicated groups was done by the Wilcoxon rank-sum test with continuity correction. (**, P < 0.01; ***, P < 0.001).

Article Snippet: The eCT26 Kras G12C /G12C Abcb1 − / − (clone I20) and eCT26 Kras G12D /G12D Abcb1 − / − (clone I12) cell lines were engineered from the murine CT26 homozygous Kras G12D tumor cell line (ATCC CRL-2638).

Techniques: In Vitro, Inhibition, Binding Assay, Recombinant, Western Blot, Mutagenesis, Labeling, Comparison

Journal: Cancer Discovery

Article Title: Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers

doi: 10.1158/2159-8290.CD-24-0027

Figure Lengend Snippet: RMC-6236 inhibits RAS signaling and tumor growth and drives tumor regressions in vivo. A, Blood and tumor PK profiles of RMC-6236 in Capan-2 ( KRAS G12V/WT , PDAC) xenograft tumor-bearing BALB/c nude mice. Tumor-bearing mice were treated with a single dose of vehicle or RMC-6236 at 3, 10, or 25 mg/kg. Blood and tumors were harvested at indicated time points ( n = 3/time point/dose). PK profiles are shown as RMC-6236 concentration in tumors (green lines) and blood (red lines) over time. Shades of green or red represent PK profiles at three tested doses. The dashed lines represent EC 50 and EC 90 potency of RMC-6236 in inhibiting DUSP6 mRNA expression in Capan-2 tumors derived from the PK/PD relationship curve in . Values are plotted as mean ± SEM. B, PD of RMC-6236 in Capan-2 ( KRAS G12V/WT , PDAC) xenograft tumors, shown as the relative change in DUSP6 mRNA expression. Tumor-bearing mice were treated with a single dose (solid lines) of vehicle, RMC-6236 at 3, 10, or 25 mg/kg, or 7 consecutive daily doses of RMC-6236 at 25 mg/kg (dashed lines). Shades of green represent three tested doses. Solid lines represent a single dose while the dashed line represents repeat dosing. Values are plotted as mean ± SEM. C, Histopathology analysis of Capan-2 xenograft tumors treated with a single dose of vehicle control, or RMC-6236 at 3, 10, or 25 mg/kg or 7 consecutive daily doses of RMC-6236 at 25 mg/kg and collected at indicated time points ( n = 2–3/time point/dose). pERK staining in tumor areas was quantified and compared with vehicle using one-way ANOVA followed by Dunnett multiple comparison test (*, P < 0.05; **, P < 0.01; *** , P < 0.001). Representative images are shown at 200× magnification from samples closest to the mean of the group. Scale bar, 50 μm. D–G, Dose-dependent antitumor activity of RMC-6236 in subcutaneous xenograft models of ( D ) Capan-2 ( KRAS G12V/WT , PDAC; n = 8 per group), po qd, per os quaqua (once a day) ( E ) NCI-H441 ( KRAS G12V/WT , NSCLC; n = 10 per group), ( F ) HPAC ( KRAS G12D/WT , PDAC; n = 10 per group), and ( G ) NCI-H358 ( KRAS G12C/WT , NSCLC; n = 8–10 per group). Tumor-bearing mice were treated with vehicle or RMC-6236 at indicated doses for 27–28 days, and mean tumor volumes of each group were plotted over the course of treatment. Vehicle control and RMC-6236 groups were compared by two-way repeated-measures ANOVA on the last measurement day of the vehicle group (***, P < 0.001). The dotted line indicates the initial average tumor volume. Error bars, SEM. # indicates 1 animal terminated upon reaching a tumor burden endpoint.

Article Snippet: The eCT26 Kras G12C /G12C Abcb1 − / − (clone I20) and eCT26 Kras G12D /G12D Abcb1 − / − (clone I12) cell lines were engineered from the murine CT26 homozygous Kras G12D tumor cell line (ATCC CRL-2638).

Techniques: In Vivo, Concentration Assay, Expressing, Derivative Assay, Histopathology, Control, Staining, Comparison, Activity Assay

Journal: Cancer Discovery

Article Title: Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers

doi: 10.1158/2159-8290.CD-24-0027

Figure Lengend Snippet: Translating RMC-6236 activity in NSCLC. A, Efficacy of RMC-6236 on Kras G12C , Kras G12D , Kras G12V , Kras G12A , Kras G13D , or Kras Q61H -driven autochthonous lung tumors in immunocompetent mice. A pool of lentiviral cDNA vectors encoding each oncogenic Kras variant was delivered intratracheally to the lungs of each mouse, and 13 weeks after tumor growth, mice were treated with RMC-6236 at 20 mg/kg po qd for 3 weeks prior to analysis. 95% confidence intervals are shown. B, Efficacy of RMC-6236 and adagrasib in the LUN055 NSCLC PDX model with KRAS G12C allele copy-number gain. Immunoblot Western analyses (left) of RAS and KRAS protein levels in NCI-H358 ( KRAS G12C/WT , NSCLC), LU99 ( KRAS G12C/WT , NSCLC), NCI-H2122 ( KRAS G12C/G12C , NSCLC), and LUN055 ( KRAS G12C/WT , NSCLC) xenograft tumors. Relative copy-number (middle) of KRAS WT or KRAS G12C in LUN055 xenograft tumors ( n = 2) were determined by ddPCR and normalized to ACTB . LUN055 xenograft tumor-bearing mice were treated with vehicle or RMC-6236 at 25 mg/kg po qd or adagrasib at 100 mg/kg po qd for 24 to 28 days ( n = 3 per group, right). Mean tumor volumes of each group were plotted over the course of treatment. Dotted line indicates the initial average tumor volume. Error bars, SEM. C, Efficacy of RMC-6236 in the intracranially implanted LU99-Luc ( KRAS G12C/WT , NSCLC) xenograft model ( n = 8 per group). RMC-6236 was dosed at 25 mg/kg daily for 21 days. Images of bioluminescence in individual mice were shown. Bioluminescence of ROI in vehicle control and RMC-6236 groups were compared by two-way repeated-measures ANOVA at day 21 (**, P < 0.01). Results were shown as mean ± SEM. D, Antitumor activity of RMC-6236 and the combination with anti–PD-1 (clone RMP1-14, rat IgG2a) following repeated administration in BALB/c mice bearing the murine colon carcinoma eCT26 ( Kras G12C/G12C ) shown as individual tumor growth curves ( n = 10 per group). Graphs indicate the number of complete regressions per injected mice. RMC-6236 and anti–PD-1 treatment started on day 17 after implantation. RMC-6236 treatment was stopped at day 31 after implantation and anti–PD-1 at day 35 after implantation. E, Antitumor activity of RMC-6236 following repeated administration in NSG mice bearing the murine colon carcinoma eCT26 ( Kras G12C/G12C ) shown as individual tumor growth curves ( n = 10 per group). Graphs indicate the number of complete regressions per injected mice. RMC-6236 treatment started on day 16 after implantation. F, Immune cell composition (CD8 + and CD4 + T cells, Ly6C + and Ly6G + myeloid-derived suppressor cells and M2 macrophages) in murine colon carcinoma eCT26 syngeneic tumors ( Kras G12C/G12C ) represented as percentage of CD45 + cells and expression of cell-surface markers on viable, CD45 − large cells (assessed as tumor cells) 24 hours post 4 days of treatment with vehicle or RMC-6236 at 25 mg/kg po qd n = 3 biological replicates/group represented as mean; *, P < 0.05; **, P < 0.01; ns, nonsignificant by two-sided Student t test.

Article Snippet: The eCT26 Kras G12C /G12C Abcb1 − / − (clone I20) and eCT26 Kras G12D /G12D Abcb1 − / − (clone I12) cell lines were engineered from the murine CT26 homozygous Kras G12D tumor cell line (ATCC CRL-2638).

Techniques: Activity Assay, Variant Assay, Western Blot, Control, Injection, Derivative Assay, Expressing

Journal: Cancer Discovery

Article Title: Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers

doi: 10.1158/2159-8290.CD-24-0027

Figure Lengend Snippet: Activity of RMC-6236 in pancreatic and lung cancer patients. A, Pretreatment and 12-week (post cycle 4) scans of a heavily pretreated patient with a KRAS G12D mutation-positive PDAC indicating a complete response of both target and nontarget lesions. Patient continued on study treatment in cycle 6. Axial views of computed tomography (CT) abdomen images prior to RMC-6236 treatment (top) and after four cycles of RMC-6236 treatment (bottom). B, Pretreatment and 6-week (post cycle 2) scans of a patient with a KRAS G12V mutation-positive NSCLC indicating a complete response of target lesions (no nontarget lesions present at baseline), atelectatic changes in the right lung are also largely resolved by 6 weeks. Complete response was confirmed at cycle 4, and the patient continued on study treatment with a complete response in cycle 10. Axial views of computed tomography (CT) chest images prior to RMC-6236 treatment (top) and after two cycles of RMC-6236 treatment (bottom).

Article Snippet: The eCT26 Kras G12C /G12C Abcb1 − / − (clone I20) and eCT26 Kras G12D /G12D Abcb1 − / − (clone I12) cell lines were engineered from the murine CT26 homozygous Kras G12D tumor cell line (ATCC CRL-2638).

Techniques: Activity Assay, Mutagenesis, Computed Tomography