Abstract
RAS family members are the most frequently mutated oncogenes in human cancers. Although KRAS(G12C)-specific inhibitors show clinical activity in patients with cancer1,2,3, there are no direct inhibitors of NRAS, HRAS or non-G12C KRAS variants. Here we uncover the requirement of the silent KRASG60G mutation for cells to produce a functional KRAS(Q61K). In the absence of this G60G mutation in KRASQ61K, a cryptic splice donor site is formed, promoting alternative splicing and premature protein termination. A G60G silent mutation eliminates the splice donor site, yielding a functional KRAS(Q61K) variant. We detected a concordance of KRASQ61K and a G60G/A59A silent mutation in three independent pan-cancer cohorts. The region around RAS Q61 is enriched in exonic splicing enhancer (ESE) motifs and we designed mutant-specific oligonucleotides to interfere with ESE-mediated splicing, rendering the RAS(Q61) protein non-functional in a mutant-selective manner. The induction of aberrant splicing by antisense oligonucleotides demonstrated therapeutic effects in vitro and in vivo. By studying the splicing necessary for a functional KRAS(Q61K), we uncover a mutant-selective treatment strategy for RASQ61 cancer and expose a mutant-specific vulnerability, which could potentially be exploited for therapy in other genetic contexts.
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Data availability
FASTQ files from the Amplicon sequencing of KRAS are available from the Sequence Read Archive database under BioProject accession number PRJNA789849. Non-synonymous and silent mutations in KRAS, NRAS and HRAS genes were obtained from TCGA pan-cancer cohort (https://portal.gdc.cancer.gov). Data on exon 3 skipping at baseline in the TCGA cohort were obtained from TCGA SpliceSeq (https://bioinformatics.mdanderson.org/public-software/tcgaspliceseq/). Gene effect scores for dependency, evaluated by RNAi and CRISPR knockout, were obtained from Depmap (https://depmap.org/portal/). Source data are provided with this paper.
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Acknowledgements
Y.K. is supported in part by JSPS Overseas Research Fellowships, Uehara Memorial Foundation, SGH Foundation and Suzuken Memorial Foundation. P.A.J. is supported in part by the Cammarata Family Foundation Research Fund, the American Cancer Society Clinical Research Professor Grant (CRP-17-111-01-CDD), the Mark Foundation for Cancer Research (grant no. 19-029 MIA), the Mock Family Fund for Lung Cancer Research and the Goldstein Family Research Fund. We thank S. Obika and T. Nakayama for advice on antisense oligonucleotides; D. A. Barbie for helpful discussion; and E. F. Cohen for visualizing fastq data on KRAS transcript reads in IVG software.
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Contributions
Y.K. conceptualized the study, collected data from TCGA and DFCI cohorts, designed antisense oligonucleotides, developed and executed in vitro experiments and wrote the paper. C.C. executed in vivo studies. J.L. executed in vitro experiments. K.S.P., L.A.K., J.L.H. and A.I.H. collected data from the Guardant Health cohort. Z.W. and F.H. performed statistical analyses. M.B. executed in vitro experiments, interpreted data, and wrote the paper. P.C.G. supervised and analysed in vivo studies. P.A.J. conducted and supervised all experiments, interpreted data and wrote the paper. All authors reviewed and commented on the paper.
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P.A.J. reports consulting fees from AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche/Genentech, Takeda Oncology, ACEA Biosciences, Eli Lilly and Company, Araxes Pharma, Ignyta, Mirati Therapeutics, Novartis, Loxo Oncology, Daiichi Sankyo, Sanofi Oncology, Voronoi, SFJ Pharmaceuticals, Silicon Therapeutics, Nuvalent, Esai, Bayer, Biocartis, Allorion Therapeutics, Accutar Biotech and AbbVie; receiving post-marketing royalties from DFCI owned intellectual property on EGFR mutations licensed to Lab Corp; sponsored research agreements with AstraZeneca, Daichi-Sankyo, PUMA, Boehringer Ingelheim, Eli Lilly and Company, Revolution Medicines and Astellas Pharmaceuticals; and stock ownership in Loxo Oncology and Gatekeeper Pharmaceuticals. K.S.P., L.A.K., J.L.H. and A.I.H. are employees and stockholders of Guardant Health. P.A.J. and Y.K. are inventors on a patent on the therapeutic use of mutant specific antisense oligonucleotides.
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Extended data figures and tables
Extended Data Fig. 1 CRISPR-Cas9 modified EGFR mutant PC-9 cells for evaluating oncogenicity of KRAS or BRAF mutations.
a, A schema of selection with EGFR inhibitor osimertinib. Majority of parental EGFR-mutant PC-9 cells are sensitive to osimertinib with only a small fraction exhibiting intrinsic resistance. In bulk CRISPR-Cas9 modified PC-9 cells, osimertinib treatment can lead to an increase in the fraction of cells harboring a resistance mutation such as KRAS G12C. b, Cell viability assay of parental and CRISPR-Cas9 modified PC-9 cells after 72 h of osimertinib treatment. Each clone has heterozygous KRAS mutations: GQ60GK c.180_181delinsCA plus Q61K, GQ60GK c.180_181delinsGA plus frameshift, and Q61K plus frameshift (n = 3 biological replicates, mean ± s.d.). c, Knockdown of KRAS or BRAF in CRISPR-Cas9-modified PC-9 clones following 48 h of KRAS or BRAF specific siRNA treatment are shown by western blot analyses.
Extended Data Fig. 2 Alternative splicing of KRAS in CRISPR-Cas9 modified PC-9 cells.
a, Images of KRAS-specific PCR amplicons of cDNA, generated from CRISPR-Cas9 modified PC-9 clones expressing different KRAS mutations in the presence or absence of osimertinib given the influence of upstream EGFR signals. Heterozygosity or homozygosity of KRAS mutants is shown for each clone. M: 100 bp-marker, m: mutant, wt: wild-type, fs: frameshift. b, Images of KRAS-specific PCR amplicons of cDNA, generated from additional CRISPR-Cas9 modified PC-9 clones expressing different KRAS mutations. c, Images of KRAS-specific PCR amplicons of cDNA, generated from KRAS mutant cell lines and CRISPR-Cas9 modified PC-9 clones expressing different KRAS mutations.
Extended Data Fig. 3 A strategy to convert the original KRAS GQ60GK into the non-functional Q61K by editing silent mutations using CRISPR-Cas9.
a, A schema of the proposed alternative treatment strategy using CRISPR-Cas9 editing with a mutant-specific sgRNA substituting the c.180 silent mutation with a cryptic splice donor nucleotide in order to promote exonic skipping leading to a premature STOP codon b, Allele frequencies of mutations, evaluated by next generation sequencing using DNA derived from bulk KRAS GQ60GK-mutant Calu6 and SNU668 cell lines, 48 h after CRISPR-Cas9 editing with indicated donor templates (n = 1). Allele frequency of original KRAS GQ60GK decreased by CRISPR editing with GQ60GK-specific sgRNA and allele frequency of non-functional Q61K increased only in the presence of donor template designed for Q61K. c, Relative expression of indicated KRAS isoforms, evaluated by qPCR in KRAS GQ60GK-mutant Calu6 and SNU668 cell lines, 48 h after CRISPR-Cas9 editing with indicated donor templates. Expression data are shown as relative to cell lines using Q61K template (n = 3 biological replicates, mean ± s.d.). Although it is difficult to capture clones successfully converted to non-functional Q61K due to their inability to grow, increased expression level of the KRAS isoform skipping 112 bp was confirmed in the remaining cells 2 days after CRISPR-editing.
Extended Data Fig. 4 Designing mutant-selective antisense oligos.
a, Structures of DNA, antisense oligo nucleotides with phosphorothioate (PS) + 2’-O-Methoxyethyl (2’MOE) modifications, and morpholino. In PS+2’MOE, a non-bridging oxygen is replaced by a sulfur atom in the phosphate backbone, and 2′ position of the sugar moiety is modified. In morpholino, the sugar moiety is replaced with methylenemorpholine rings, and the anionic phosphates are replaced with non-ionic phosphorodiamidate linkages. b, Schema depicting the design of the KRAS GQ60GK c.180_181delinsCA, NRAS Q61K, and HRAS Q61L selective antisense oligo. The motifs for binding exonic splicing enhancers simulated using the Human Splicing Finder (top) and ESE finder (bottom) are shown. Matrices for SR proteins including SRSF1 (SF2/ASF and IgM-BRCA1), SRSF2 (SC35), SRSF5 (SRp40), and SRSF6 (SRp55) are shown.
Extended Data Fig. 5 Mutant selective inhibition of RAS using morpholino.
Raw NGS reads showing KRAS Q61L transcript were visualized using IGV 2.8.0 software.
Extended Data Fig. 6 In vitro sensitivity to MEK inhibitor and morpholino oligos in RAS mutant cells.
a. Cell viability assays of suspension cells after 8 days of 10 μM morpholino treatment were performed on ultra-low attachment plates (n = 6 biological replicates, mean ± s.d., ANOVA, followed by Dunnett’s post-hoc test comparing to cells treated with DMSO, **p < 0.01). b, Cell viability assay of a panel of mutant RAS cell lines after 72 h of trametinib treatment in 2D adherent or 3D suspension culture (n = 3 biological replicates, mean ± s.d.). c, The correlation of growth inhibition by 10 nM trametinib in 2D or 3D culture and morpholino antisense oligo nucleotide in 17 RAS Q61X cell lines. d, Western blot analyses of signaling in KRAS wild-type cell lines were performed after 72 h treatment with 10nM trametinib, 10 μM morpholino or respective controls. e, Relative expression of ERK signature genes, evaluated by qPCR in Calu6 and H650 cell lines treated with mutant-selective morpholino for 48 h. Expression data are normalized to readout of a control morpholino treatment. GUSB was used as an internal control. n = 3 biological replicates, mean ± s.d., t test, **p < 0.01. f, Cell viability assays in suspension cells after 8 days of 50 nM afatinib, 10μg/ml cetuximub, and 10 μM morpholino treatment were performed with the same method as a (n = 6 biological replicates, mean ± s.d., ANOVA, followed by Dunnett’s post-hoc test comparing to cells treated with DMSO, **p < 0.01). g, Western blot analyses of signaling in NRAS and HRAS mutant cell lines were performed after 72 h of treatment with 10 nM trametinib, 10 μM morpholino, or DMSO.
Extended Data Fig. 7 Pre-treatment strategy using vivo-morpholino in vitro and in vivo H650 models.
a, Morpholino oligo pre-treatment strategy. H650 cells were pre-treated with 10 μM control vivo-morpholino and target vivo-morpholino for 1 to 4 days as 3D suspension cells. After drug washout, cells were cultured in growth media and cell viability was evaluated on day 8 in vitro. Luciferase-expressing H650 cells were used for in vivo experiments. After pre-treatment with 10 μM vivoMor-CTRL and target vivo-morpholino for 1 to 2 days as 3D suspension cells, drugs were washed out and cells were subcutaneously implanted into mice. In vivo imaging was performed twice a week. b, In vitro cell viability assays of H650 cells pre-treated with 10μM vivoMor-CTRL or vivoMor-4 for 1 to 4 days (n = 6 biological replicates, mean ± s.d., t test, **p < 0.01). c, Volume change of pre-treated H650 xenograft tumors. H650 cells were pre-treated with vivoMor-CTRL or vivoMor-4 in vitro for 1 or 2 days prior to injection into nude mice (n = 10 per each group, mean ± s.e.m., t-test and linear mixed growth models at day 22, **p < 0.01).
Extended Data Fig. 8 Intra-tumoral injection of vivo-morpholino in H650 xenograft models.
a, Images of KRAS-specific PCR amplicons generated from the cDNA of H650 xenograft tumors that were treated with daily intra-tumoral injection of morpholino for 7days. Fraction of exon 3 skipping is defined as the band intensities of “skipped/(skipped + full-length)” as measured by ImageJ (n = 1). M: 100 bp-marker. b, Fractions of exon 3 skipping in samples shown in (a) were compared using t test, **p < 0.01, n = 2–6 mice in each group, box plots show minimum, lower quartile, median, upper quartile, and maximum. c, Relative expression of KRAS exon 3 skipping, evaluated by qPCR in tumor samples corresponding to (a) (n = 3 biological replicates, mean ± s.e.m.). d, Body weight change of mice with H650 xenograft tumors treated with morpholino over time (n = 10 per each group, mean ± s.e.m.).
Extended Data Fig. 9 Pre-treatment strategy and intra-tumoral injection of vivo-morpholino in Calu6 models.
a, In vitro cell viability assays of Calu6 cells pre-treated with 10 μM vivoMor-CTRL or vivoMor-1 for 1 to 4 days (n = 6 biological replicates, mean ± s.d., t test, **p < 0.01). b, Volume change of pre-treated Calu6 xenograft tumors. Calu6 cells were pre-treated with vivoMor-CTRL or vivoMor-1 in vitro for 24 h prior to injection into nude mice (n = 10 per each group, mean ± s.e.m., t-test and linear mixed growth models, *p < 0.05, **p < 0.01). c, In vivo efficacy of Calu6 xenograft tumors treated with daily intra-tumoral injection of morpholino (n = 10 per each group, mean ± s.e.m., t-test and linear mixed growth models, **p < 0.01). d, Body weight change of mice with Calu6 xenograft tumors treated with morpholino over time (n = 10 per each group, mean ± s.e.m.).
Extended Data Fig. 10 Mutant selective inhibition of KRAS using PS+2’MOE antisense oligos.
a, Schema depicting the design of the KRAS GQ60GK c.180_181delinsCA selective antisense oligo by screening. b, Images of KRAS-specific PCR amplicons generated from the cDNA of cells treated with 6 kinds of PS+2’MOE antisense oligos for 48 h. Exon 3 skipping fraction is defined as the band intensities of “skipped/(skipped + full-length)” transcript as quantified by ImageJ. M: 100 bp-marker. n = 2 biological replicates, mean ± s.e.m. c, Images of KRAS-specific PCR amplicons in indicated cell lines with same method as b (n = 2 biological replicates, mean ± s.e.m.). d, Transcript reads of KRAS GQ60GK or Q61L versus wild-type in the intact full-length KRAS amplicon derived from mRNA of Calu6 and SW948 cells treated with PS+2’MOE antisense oligos (n = 1). e, Western blot analyses of signaling in KRAS mutant and wild-type cell lines were performed after 6 days of 0.5 μM PS+2’MOE antisense oligos. f, Cell viability assays in suspension cells after 8 days of 0.5 μM PS+2’MOE antisense oligos treatment were performed (n = 6 biological replicates, mean ± s.d., t test, **p < 0.01).
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Kobayashi, Y., Chhoeu, C., Li, J. et al. Silent mutations reveal therapeutic vulnerability in RAS Q61 cancers. Nature 603, 335–342 (2022). https://doi.org/10.1038/s41586-022-04451-4
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DOI: https://doi.org/10.1038/s41586-022-04451-4
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