Phase and context shape the function of composite oncogenic mutations

Abstract

Cancers develop as a result of driver mutations1,2 that lead to clonal outgrowth and the evolution of disease3,4. The discovery and functional characterization of individual driver mutations are central aims of cancer research, and have elucidated myriad phenotypes5 and therapeutic vulnerabilities6. However, the serial genetic evolution of mutant cancer genes7,8 and the allelic context in which they arise is poorly understood in both common and rare cancer genes and tumour types. Here we find that nearly one in four human tumours contains a composite mutation of a cancer-associated gene, defined as two or more nonsynonymous somatic mutations in the same gene and tumour. Composite mutations are enriched in specific genes, have an elevated rate of use of less-common hotspot mutations acquired in a chronology driven in part by oncogenic fitness, and arise in an allelic configuration that reflects context-specific selective pressures. cis-acting composite mutations are hypermorphic in some genes in which dosage effects predominate (such as TERT), whereas they lead to selection of function in other genes (such as TP53). Collectively, composite mutations are driver alterations that arise from context- and allele-specific selective pressures that are dependent in part on gene and mutation function, and which lead to complex—often neomorphic—functions of biological and therapeutic importance.

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Fig. 1: Composite mutations in human cancers.
Fig. 2: Gene- and residue-specific selective pressure for composite mutations.
Fig. 3: cis- and trans-acting composite mutants.
Fig. 4: Mutant-allele-specific enrichment for composite mutations.

Data availability

All mutational data from the prospective sequencing cohort are available at http://download.cbioportal.org/composite_mutations_maf.txt.gz. Mutational data from The Cancer Genome Atlas were acquired from https://gdc.cancer.gov/about-data/publications/pancanatlas. RNA sequencing data have been deposited in the Gene Expression Omnibus with accession number GSE136295. All other genomic and clinical data accompany the Article, and are available in the Extended Data and Supplementary Information. All other materials are available upon request from the corresponding authors.

Code availability

Source code for these analyses is available at https://github.com/taylor-lab/composite-mutations.

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Acknowledgements

We thank the members of the E.R. and B.S.T. laboratories for discussion and support. This work was supported by National Institutes of Health awards P30 CA008748, P01 CA087497 (S.W.L.), U54 OD020355 (S.W.L. and B.S.T.), R01 CA207244 (B.S.T.), R01 CA204749 (B.S.T.), R01 CA245069 (B.S.T.); Brown Performance Group ICI Fund (N.V. and E.R.), Society of MSK (N.V. and E.R.), American Cancer Society, Anna Fuller Fund and the Josie Robertson Foundation (B.S.T.). F.J.S.-R. is an HHMI Hanna Gray Fellow supported in part by an MSKCC Translational Research Oncology Training Fellowship (T32-CA160001). S.W.L. is an investigator of the Howard Hughes Medical Institute.

Author information

Affiliations

Authors

Contributions

A.N.G., E.R. and B.S.T. conceived the study. C.M.B., E.B., P.J., A.V.P., A.L.R., N.D.F., C.B., N.S., E.R. and B.S.T. assisted with genomic data collection and analytical methodology development. F.J.S.-R., Y.C., N.V., M.S. and S.W.L. designed and performed the experiments. Y.J.H. and T.B. assisted with RNA sequencing. A.N.G., E.R. and B.S.T. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Ed Reznik or Barry S. Taylor.

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Competing interests

N.V. reports advisory board activities for Novartis and consulting activities for Petra Pharmaceuticals. M.S. has received research funding from Puma Biotechnology, Daiichi-Sankio, Immunomedics, Targimmune and Menarini Ricerche; is a cofounder of Medendi.org, and is on the advisory boards of the Bioscience Institute and Menarini Ricerche. S.W.L. is a founder and scientific advisory board member of Oric Pharmaceuticals, Mirimus, Inc. and Blueprint Medicines; and is on the scientific advisory boards of Constellation Pharmaceuticals, Petra Pharmaceuticals and PMV Pharmaceuticals. B.S.T. reports receiving honoria and research funding from Genentech and Illumina, and advisory board activities for Boehringer Ingelheim and Loxo Oncology, a wholly owned subsidiary of Eli Lilly, Inc. All stated activities were outside of the work described here. The other authors declare no competing interests.

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Peer review information Nature thanks Moritz Gerstung, Mark Lackner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Study cohort and rates of composite mutations.

a, Distribution of cancer types in the study cohort. b, The rate of composite mutations (22.7% of all tumours) compared to a simulated background rate (black, P = 10−5 from one-sided permutation test for enrichment with 100,000 random permutation-based simulations (no permutation exceeded observed value)). c, The observed rate of composite mutations in the primary untreated cancers of the TCGA cohort (n = 10,908 solid tumours) when controlling for gene content for consistency with the targeted sequencing panel of the prospective cohort studied here. The null distribution from sampling (Methods) is shown in black. d, The observed and expected rate of composite mutations in tumours of the indicated tumour mutational burden (as in Fig. 1b, n = 30,505 biologically independent tumour samples with tumour mutational burden ≤ 40, P = 1 × 10−9 from two-sided Wilcoxon signed-rank test).

Extended Data Fig. 2 Sources of local hypermutation.

a, The number of composite mutations comprising two or more constituent variants (top) and the distribution of likely causative mutational signatures among them (bottom). Composite mutants comprising greater than three mutations were increasingly produced by APOBEC-associated mutagenesis, indicative of localized hypermutation53,54, but accounted for a minority of events cohort-wide. b, Left, the somatic mutational data in the study cohort reflect the elevated mutation rates previously observed at both the positions closest to the nucleosome dyad as well as DNA bound to active transcription-factor binding sites38,39. However, mutations arising in composite events were proportionally less often proximal to such sites (defined here as within the full width at half maximum of the peak of mutation rate (red)) than were singleton mutations (right, P = 10−27 and 10−47, respectively; two-sided two-sample Z-test, n = 323,883 single-nucleotide substitutions arising in 471 biologically distinct melanoma samples).

Extended Data Fig. 3 Number and distribution of composite events across genes.

a, The number and percentage of cases in the study cohort containing composite mutations in the indicated genes (right) juxtaposed to their overall mutation rate (left). Genes with a significant enrichment of composite mutations are shown (Q < 0.01, FDR-adjusted P values from one-sided binomial test for enrichment, n = 26,997 as in Fig. 2b), limited to the top 10 genes by significance in each category of gene function, unless fewer. b, The significance of enrichment for composite mutations (n and statistical tests as described in a and Fig. 2b) limited to 168 oncogenes.

Extended Data Fig. 4 cis composite secondary-resistance mutations.

The cis composite mutations classified as arising in post-treatment specimens due to acquired resistance to one of several molecularly targeted therapies in the study cohort.

Extended Data Fig. 5 Phenotypic characterization of TP53 composite mutants.

a, TP53R280T/E287D mutant lung adenocarcinoma. Left, mutant allele fractions of clonal TP53 mutations consistent with loss of wild-type TP53 (error bars, 95% binomial confidence intervals). Expected mutant allele fractions of different copy number states are shown as horizontal lines. Mutant KEAP1 in the same tumour (with LOH) is shown for reference. Right, spanning reads indicating cis mutations. b, Right and left, Trp53 and Cdkn1a mRNA expression in KrasG12D/+Trp53Mut mouse lung cancer cells expressing distinct Trp53 genotypes. Bars, average of three replicates, error bars are 95% confidence intervals. c, The aggregate Z-score per replicate for the mRNA expression of canonical p53-target genes (n = 3 replicates per allele; box centre is median, edges are 25% and 75% quartiles, whiskers are minimum and maximum of the most extreme values). d, Principal component analysis of the transcriptomes of Trp53 genotypes (n = 3 replicates shown per condition). e, Dendrogram as in Fig. 3f, indicating the genes of interest (effectors of the AP-1 transcription factor network (PID_AP1_PATHWAY; Q = 1.4 × 10−7 based on computed overlap (using mSigDB) with n = 5,501 gene sets from the curated C2 collection)). f, The prevalence of TP53R280T and TP53E287D mutations (top), and the fraction arising as composite mutants (bottom). The corresponding mouse alleles are given in parentheses. g, Principal component analysis of the transcriptomes of the Trp53R277K/E282K composite mutation genotypes (as in d). n = 3 replicates per allele. h, The percentage of GFP+ FACS-purified KrasG12D/+Trp53−/− lung adenocarcinoma cells stably transduced with pMIG-empty or pMIG-p53-R277T-E284D, and cultured in vitro for 10 days in a 60:40 mixture with untransduced parental cells. Bar indicates mean, error bars are s.d., n = 3 independent infections. i, Overall survival of immunocompromised mice bearing lung tumours of the indicated Trp53 genotypes generated by tail vein injection of stably transduced and FACS-purified KrasG12D/+Trp53−/− lung adenocarcinoma cells (n = 100,000 cells).

Extended Data Fig. 6 Saturation analysis of genes for composite mutation detection.

Down-sampling indicates the number of residues identified as enriched for arising in composite mutations in each of four genes (Q < 0.1, FDR-adjusted one-sided Fisher’s exact tests as in Fig. 4a; n = 1,000–26,997 patients per down-sample) as a function of the number of tumours sequenced (LOESS fit is shown with 95% confidence interval). Four genes that accounted for the greatest proportion of all enriched residues detected are shown (Fig. 4a). EGFR appears to reach saturation for discovery of residues enriched for arising in composite, whereas the other genes have not yet reached saturation for discovery at the current cohort size.

Extended Data Fig. 7 Mutational signature attribution among composite mutations.

a, The fraction of all composite mutations identified here in which one or both individual mutations could be unambiguously attributed to an established mutational signature. The majority of composite variants could not be directly attributed to APOBEC, ultraviolet, smoking or other known mutational signatures. b, The fraction of composite mutations per gene in which one or both variants could be attributed to an established mutational signature.

Extended Data Fig. 8 Conditional mutant alleles.

a, The number of affected cases containing each of the indicated somatic mutations in TERT, EGFR or PIK3CA as either individual mutations (top) or as part of composite mutants (bottom). Conditional mutations were defined as those statistically enriched for arising as part of composite mutations, but seldom as individual hotspot mutations in cancer (predominantly accompanied by a second somatic mutation). b, The incidence of TERT promoter mutations and the fraction arising as composite mutations (orange). Bottom, the co-occurrence and mutual exclusivity of composite mutations in the TERT promoter (The P values for n = 5 and 6 co-occurring mutations are 0.002 and 3 × 10−7, respectively, and for 0 mutually exclusive mutations is 1 × 10−25; two-sided Fisher’s exact test, n = 29,507 patients). c, Transcription factor GABPA binding affinity for mutant and wild-type TERT promoter sequences at the 228G>A, 250G>A and the conditional 205G>A allele.

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Gorelick, A.N., Sánchez-Rivera, F.J., Cai, Y. et al. Phase and context shape the function of composite oncogenic mutations. Nature 582, 100–103 (2020). https://doi.org/10.1038/s41586-020-2315-8

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