KRASG12C selective inhibitors, such as sotorasib and adagrasib, have raised hopes of targeting other KRAS-mutant alleles in patients with cancer. We report that KRAS wild-type (WT)–amplified tumor models are sensitive to treatment with the small-molecule KRAS inhibitors BI-2493 and BI-2865. These pan-KRAS inhibitors directly target the “OFF” state of KRAS and result in potent antitumor activity in preclinical models of cancers driven by KRAS-mutant proteins. In this study, we used the high-throughput cellular viability Profiling Relative Inhibition Simultaneously in Mixtures assay to assess the antiproliferative activity of BI-2493 in a 900+ cancer cell line panel, expanding on our previous work. KRAS WT–amplified cancer cell lines, with a copy number >7, were identified as the most sensitive, across cell lines with any KRAS alterations, to our pan-KRAS inhibitors. Importantly, our data suggest that a KRAS “OFF” inhibitor is better suited to treat KRAS WT–amplified tumors than a KRAS “ON” inhibitor. KRAS WT amplification is common in patients with gastroesophageal cancers in which it has been shown to act as a unique cancer driver with little overlap to other actionable mutations. The pan-KRAS inhibitors BI-2493 and BI-2865 show potent antitumor activity in vitro and in vivo in KRAS WT–amplified cell lines from this and other tumor types. In conclusion, this is the first study to demonstrate that direct pharmacologic inhibition of KRAS shows antitumor activity in preclinical models of cancer with KRAS WT amplification, suggesting a novel therapeutic concept for patients with cancers bearing this KRAS alteration.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
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Supplementary data

Supplementary Tables 1-5

Supplementary Table 1: PRISM data integrated with omics data from depmap Supplementary Table 2:Top 20 sensitive cell lines (PRISM) Supplementary Table 3:Frequency of KRAS WT AMP in AACR Genie v 15.1 (MSK cohort) Supplementary Table 4: Mututal exclusivity for KRAS WT AMP (results downloaded from AACR Genie v15.1) Supplementary Table 5 IC50 values from CTG assays for KRAS wt amplified cell lines

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Supplementary Figure 1

Supplementary Figure 1: Cellular sensitivity to BI-2493 integrated with CRISPR and RNAi gene dependency data. (Top) CRISPR: Drug-target associations show selectivity of BI-2493 for KRAS but not for HRAS and NRAS. Panel left, mid and right show gene effect (or dependency) scores derived from Chronos for KRAS, HRAS and NRAS, respectively on the x-axis. A low gene effect score indicates that a cell line is likely to depend on a given gene. A score equal to or close to 0 indicates genes that are non-essential, whereas a score of -1 is defined as the median of all common essential genes and is commonly used as a threshold for gene dependency (indicated by a red dotted vertical line). The y-axis shows drug sensitivity values reported as 1-AUC for BI-2493 derived from the PRISM screen. Larger values indicate higher sensitivity. KRAS Pearson R=-0.385, P=2.23e-23; HRAS Pearson R=0.079, P=0.0483; NRAS Pearson R=0.184, P=3.69e-06; (Bottom) RNAi: Drug-target associations show selectivity of BI-2493 for KRAS but not for HRAS and NRAS. Panel left, mid and right show gene effect (or dependency) scores derived from Demeter2 for KRAS, HRAS and NRAS, respectively on the x-axis. KRAS Pearson R=-0.393, P=2.44e-18; HRAS Pearson R=-0.0015, P=0.9; NRAS Pearson R=0.0698, P=0.14.

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Supplementary Figure 2

Supplementary Figure 2: KRAS expression as a function of KRAS copy number alteration. (Left) KRAS expression increases with copy number. (Mid) High correlation of KRAS expression and copy number (Pearson R=0.904, P=4.85e-20). The vertical dotted line marks a relative copy number threshold of 7. Only cell lines with a relative copy number of >2 are shown. (Right) Ranking of relative copy number across all 800 cell lines from the PRISM screen. Vertical dotted lines from left to right mark relative copy number thresholds 10, 7, 2 and 1, respectively.

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Supplementary Figure 3

Supplementary Figure 3. Relationship between KRAS wild-type amplification and KRAS oncogenic activity in cell lines. RAS activation signatures (1,3) MSigDB, KrasLA, KRASG13D134, HRAS, MPAS, ras84 and RAS addiction in KRAS wild-type amplified cell lines (relative copy number >7) compared to cell lines with a lower relative copy number (2-7) and KRAS mutated cell lines without KRAS amplification. Enrichment scores were estimated using single sample enrichment (ssGSEA). A one-sided Wilcox-test was used to test for significance between KRAS relative copy number of 2-7 or >7. Adjusted P-values (Benjamini-Hochberg) are displayed.

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Supplementary Figure 4

Supplementary Figure 4. Correlation between signatures for KRAS activation and sensitivity to BI-2493 across the KRAS wild-type amplified cell panel. RAS activation signatures were obtained from East et al. (1). Correlation coefficient between enrichment scores and sensitivity to BI-2493 were estimated using a Pearson R.

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Supplementary Figure 5

Supplementary Figure 5. Anti-proliferative activity of RMC-7977 across different KRAS altered cell lines. (Left) Anti-proliferative activity of RMC-7977 (2) across different KRAS mutant or KRAS wild-type amplified cell lines. Cell lines are sorted by median sensitivity across KRAS alleles. Note: AUC values are relative measures of drug sensitivity and are therefore suitable to compare drug sensitivity across cell lines for a single compound but do not allow for a comparison across compounds. (Right) Comparison of sensitivity values for RMC-7977 (2) for cell lines with dependency on either KRAS, HRAS or NRAS. Cell lines with a Chronos score (gene effect score) of less than -1 were considered dependent. Sensitivity means between groups were tested for significance using a one-sided Wilcoxon-test.

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Supplementary Figure 6

Supplementary Figure 6: Relationship between KRAS wild-type amplification and KRAS oncogenic activity in TCGA patient data. RAS activation signatures MPAS (3), RAS_addiction and Ras84 (1). Enrichment scores were estimated using single sample enrichment (ssGSEA) in TCGA patient data. A one-sided Wilcox-test was used to test for significance between KRAS relative copy number of 2-7 or >7.

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Supplementary Figure 7

Supplementary Figure 7. Gastroesophageal cancers are enriched in KRAS wild-typeWT amplified tumors. Gastroesophageal cancers (n=3464) were selected from the AACR Genie MSK and DFCI cohort (version v16.0-public). Only patients with any alterations in the above listed genes are shown (1498 unaltered patients are not shown). Co-alterations are ranked by frequency. KRAS amplified samples are defined with a GISTIC score of 2 according to AACR GENIE as exact copy number thresholds are not available from the AACR GENIE cohort.

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Supplementary Figure 8

Supplementary Figure 8: KRAS wild-type amplified cancer cell lines are sensitive to pan-KRAS inhibitors BI-2493 and BI-2865. (A) Inhibition of pERK by BI-2493, BI-2865 and trametinib at the indicated timepoints and for the indicated cell lines (n=2, means ± SD). Control cell lines, cell lines with KRAS wild-type CN<7 and cell lines with KRAS wild-type CN>7 are colored in black, blue, and red, respectively. (B) Quantification of down regulation of pERK from (A) at 1M concentration of BI-2493 and BI-2865. P-values were calculated using two-way ANOVA, followed by Tukey’s multiple comparisons test.

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Supplementary Figure 9

Supplementary Figure 9: BI-2493 and BI-2865 treatment induces cell cycle arrest and apoptosis in KRAS wild-type amplified cancer cell lines. (A) Upper panel: Representative flow blots of cell cycle states determined by EdU incorporation into newly synthesized DNA and total DNA content staining by FxCycle of indicated cell lines treated for 48 h with DMSO (mock), 3 µM BI-2493, 3 µM BI-2865 and 0.3 µM trametinib. Cells were pre-gated based on scattering properties and DNA content. Numbers indicate frequency of parent population. Lower panel: Impact of 48 h treatment with DMSO (mock), BI-2493, BI-2865 or trametinib at the indicated concentrations on cell cycle states of the indicated cell lines (N=3 (assay was run in triplicates), means + SD) (B) Upper panel: Representative flow blots of induction of apoptosis after 48 h of treatment of the indicated cell lines with DMSO (mock), 3 µM BI-2493, 3 µM BI-2865, 0.3 µM trametinib and 2 µM Camptothecin. Cells were pre-gated based on their scattering properties. Apoptotic cells were defined by loss of inner mitochondrial membrane potential (Δψm) and detection of phosphatidylserine by Annexin V staining. Numbers indicate frequency of parent population. Lower panel: Induction of apoptosis in the indicated cell lines by treatment with DMSO (mock), BI-2493, BI-2865, trametinib or Camptothecin at the indicated concentrations after 24 and 48 h determined by flow cytometry (N=3 (assay was run in triplicates), means +SD).

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Supplementary Figure 10

Supplementary Figure 10. BI-2493 treatment in animal models is tolerated. % bodyweight change in xenograft models treated with control vehicle or BI-2493. Data represent the mean % bodyweight change +/- SEM of mice grafted with: (A) DMS 53 cells (N=7); (B) MKN1 cells (N=7). One animal in the BI-2493 treated group had to be sacrificed earlier (d11) due to bodyweight loss. (C) ES11082 PDX model (N=8). Two animals in the control vehicle treated group and three animals in the BI-2493 treated group had to be sacrificed earlier (d18, d18, d15, d12, d7, respectively). (D) GA6871 PDX model (N=8). One animal in the control vehicle treated group and 2 animals in the BI-2493 treated group had to be sacrificed earlier (d7 and d16, d30, respectively) due to bodyweight loss.

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