Fig 2: (A) K‐Ras gene copy number of BTC cell lines determined by quantitative PCR analysis of genomic DNA. (B) Representative FISH images of K‐Ras amplification status in SNU‐245 and TFK‐1 cells. CEP12 (red), K‐Ras (green) and DAPI (blue). (C) Ras activity of cell lines separated according to K‐Ras mutation and amplification status. Ras activity was assessed on cell lysates prepared from exponentially growing cells. *P < 0.05. (D) BTC cell lines were separated according to K‐Ras mutation and/or amplification status and sensitivity to everolimus at the 5, 10 and 50 nm doses compared using an unpaired t‐test.

Fig 3: Conditional KrasLSL alleles with different oncogenic mutations and codon usage.(A) Schematic of generating and activating KrasLSL alleles with the first three coding exons fused and encoded by native (nat) versus common (com) codons with either a G12D or Q61R mutation. (B) PCR genotyping of two independently derived mouse embryonic fibroblast (MEF) cultures (two biological replicates) with the indicated Kras alleles in the absence and presence of Cre recombinase (CRE) to detect the unaltered wild-type Kras allele product (WT, 488 bp) and the unrecombined (KrasLSL*, 389 bp) and recombined (LoxP recombined, 616 bp) KrasLSL allelic products. Gel images were cropped and color inverted for optimal visualization. Full-length gel images are provided in Figure 1—source data 2. (C) Expression levels, determined by immunoblot with an anti-Kras antibody, RAS activity levels, determined by RBD pull-down (RBD-PD) of lysates collected from MEF cells derived from mice with the indicated Kras alleles in the presence of Cre recombinase (CRE). MEF cultures derived from KrasLSL-natG12D/+ mice in the absence of Cre recombinase were used as negative control. MEF cultures were either serum starved overnight (starved) or serum starved overnight followed by serum stimulation for 5 min (stimulated). 20% of the elute from RBD-PD and 30 µg total protein from the total cell lysates were loaded. Tubulin serves as loading control. One of two biological replicates; see Figure 1—figure supplement 3 for the second biological replicate. Full-length gel images are provided in Figure 1—source data 3. Figure 1—source data 1.Full-length gel images of RBD pull downs.Full-length gel images from RAF1-RBD pull downs (top) and whole-cell lysates (bottom) from HEK-HTs ectopically expressing engineered Kras constructs shown in Figure 1—figure supplement 2. Figure 1—source data 2.Full-length gel image of genotyping of mouse embryonic fibroblast (MEF) cultures derived from the KrasLSL alleles in Figure 1B.PCR genotyping of two independently derived MEF cultures with the indicated KrasLSL alleles in the absence and presence of Cre recombinase (CRE) to detect the unaltered wild-type Kras allele product (WT, 488 bp) as well as the unrecombined (KrasLSL, 389 bp) and recombined (LoxP recombined, 616 bp) Kras allelic products. Gel images were color inverted for better visualization. Red box depicts region shown in Figure 1B. Figure 1—source data 3.Full-length gel images of RBD pull-downs in MEFs.Full-length gel images from RBD pull-downs (left) and whole-cell lysates (right) from MEFs derived from KrasLSL alleles with serum starvation, or serum starvation followed by serum stimulation shown in Figure 1C, and same conditions with a second clone of MEF cultures with serial dilutions of 500 µg lysate (left) and 200 µg lysate (right) used for RBD-PD as shown in Figure 1—figure supplement 3. Red box depicts regions shown in Figure 1C and Figure 1—figure supplement 3. Figure 1—source data 4.Full-length gel images of Kras expression and activity using RBD pull-downs from lung tissue.Full-length gel images from RAF1-RBD pull-downs (RBD-PD, top) and whole-cell lysates (bottom) of lungs from mice with KrasLSL alleles seven days after tamoxifen injection as shown in Figure 1—figure supplement 4A. Immunoblots of two separate pull downs from two biological replicates are shown as in Figure 1—figure supplement 4B. Red box depicts the regions shown in Figure 1—figure supplement 4B. Figure 1—source data 5.Ct values from the qRT-PCR analysis in Figure 1—figure supplement 5. Figure 1—source data 6.Sequence of coding exons 1 to 3 of the four KrasLSL alleles.Sequence alignment of the codons in the coding exons 1 to 3 of the indicated KrasLSL alleles in comparison to the murine wild-type sequence (Kras). Top: amino acid sequence. Red nucleotides: optimized codons. Green highlight: G12D mutation. Blue highlight: Q61R mutation.

Fig 4: TE of D3S-001 is insusceptible to EGF stimulation. A, Kinetics of cellular active RAS depletion in NCI-H358 cells after treatment with ARS-853 or ARS-1620 at 1 μmol/L, sotorasib, adagrasib, or D3S-001 at 100 nmol/L, in the absence (solid line) or presence (dotted line) of 40 ng/mL EGF was determined by RAS-GTP ELISA assay. B, EGF treatment stimulates the transition of RAS to GTP-bound form. NCI-H358 cells were treated with 40 ng/mL EGF at different times as indicated. Cell extracts were prepared and subjected to (left) pull-down assay and (right) pERK HTRF assay at the indicated time post-treatment. For the pull-down assay, the levels of active KRAS were quantified by chemiluminescence intensity and normalized to vehicle. For pERK HTRF, the levels of pERK were quantified by HTRF and normalized to vehicle. C, NCI-H358 cells were treated with D3S-001, sotorasib, or adagrasib with or without concurrent EGF stimulation (40 ng/mL; top left), HGF stimulation (40 ng/mL; top middle), at 100 nmol/L for 2 hours, or with or without concurrent EGF stimulation (40 ng/mL) and KRAS G12C inhibitors at their corresponding [I]50 concentrations for 1 hour (top right). Extracted cell lysates were subjected to pull-down assay to determine the effect on active KRAS. The levels of active KRAS were quantified by chemiluminescence intensity and normalized to DMSO (bottom). D, NCI-H358 cells were treated with EGF (40 ng/mL) with or without concurrent D3S-001, sotorasib, or adagrasib at their corresponding 5 × [I]50 concentrations for a time course as indicated. Cell extracts were subjected to pERK HTRF assays, and the levels of pERK were quantified by HTRF and normalized to vehicle.

Fig 5: Activity of D3S-001 in patients with NSCLC. A, The top shows baseline and C3D1 scans of a pretreated NSCLC patient with a KRASG12C mutation indicating a 47.4% reduction of a target lesion (liver metastasis). Partial response was confirmed on subsequent scans. The patient has been on treatment for more than 16 months as of March 2024 with intrapatient dose escalation in cycle 11 to 100 mg daily then to 200 mg daily in cycle 16. The patient is currently maintaining PR (target lesion decrease of −86.8%) at 400 mg daily from cycle 19. The bottom shows CT scans of the brain at baseline and at C3D1. A decreased metastatic lesion (nontarget lesion) in the cerebellum was observed. B, Baseline, C1D8, and C7D1 ctDNA of the patient. KRAS VAF of 8.76% at baseline decreased to nondetectable at C1D8 and remained undetected at C7D1. C, Plasma PK of the patient on C1D1 (day 1) and C2D1 (steady state). D, The top shows baseline and C3D1 scans of a pretreated NSCLC patient with a target lesion in the left hilar lymph node. The bottom shows baseline and C3D1 scans of another target lesion in the left axilla lymph node, indicating an overall 49% reduction of a target lesion (liver metastasis). Partial response was confirmed on subsequent scans. The patient has been on treatment for a total of 11.1 months with intra-patient dose escalation to 100 mg daily in cycle 10, then to 200 mg daily in cycle 15. The patient experienced disease progression after 8.3 months. E, Baseline, C1D8, and C7D1 ctDNA of the patient. KRAS VAF of 6.49% at baseline decreased to 2.47% at C1D8 and to undetected at C4D1. F, Plasma PK of the patient on C1D1 (day 1) and C2D1 (steady state).