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MOV file 697K, Cell undergoing a segregation error followed by cell death in the daughter cell (see Supplementary Figure 4 for movie stills)

PDF file 80K, Supplementary Figure 2: A-C) Growth curves of diploid and tetraploid clones at different passages. D) HCT-116_MLH clones DNA profiles and E) cell-to-cell variation in chromosome number using clonal FISH in HCT-116 _MLH clones

MOV file 1064K, Cell undergoing a normal mitosis followed by cell cell cycle arrest in the daughter cell (see Supplementary Figure 4 for movie stills)

Assessing APOBEC3 gene expression using immunohistochemical and bioinformatic approaches.

PDF file 45K, Supplementary Figure 8: A) Full survival curves for TCGA cohort and B) full survival curves for the validation cohort. C) Diploid only tumour survival curves from validation cohort

APOBEC3 gene expression during senescence and cell cycle progression.

PDF file 41K, Supplementary Figure 3: A-B) Segregation error type analysis in diploid and tetraploid clones. C) Colony-to-colony variation in chromosome number for HCT-116_MLH clones

PDF file 701K,Supplementary Figure 4: A-F) Still images from Supplementary Movies A-F. G) Individual clone cell-fate graph for live-cell imaging analysis and full table of numbers of cells analysed

XLS file 33K, Supplementary Table 2: Univariate and Multivariate survival analysis for TCGA and validation cohort

PDF file 106K, Supplementary Figure 5: DNA ploidy analysis of diploid and tetraploid clones at different passages

Supplementary Table 1: Overview of data resource. Supplementary Table 2: List of antibodies used for immunofluorescence. Supplementary Table 3: List of antibodies used for western blotting. Supplementary Table 4. List of primers used for PCR.

Generation and characterization of A3A and A3B knockouts in TIIP cells.

PDF file 1328K, Supplementary Figure 7: Correlation of copy number losses and gains across whole genome with increasing wGII in TCGA CRC tumours

PDF file 46K, Supplementary Table 1: List of genes on chromosome 4q recurrently lost in tetraploid clones

A complete list of investigators in the Tracking Non-Small Cell Lung Cancer Evolution through Therapy (TRACERx) Consortium

Correlation between APOBEC3 expression and different measures of CIN.

PDF file 393K,Supplementary Figure 1: A) Relationship of wGII and ploidy across cancer types. B) p53 and p21 up-regulation in response to DNA damage in diploid and tetraploid clones

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non–small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution.Significance:This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355

MOV file 1084K, Cell undergoing a normal mitosis followed by mitosis in the daughter cell (see Supplementary Figure 4 for movie stills)

Environmental carcinogenic exposures are major contributors to global disease burden yet how they promote cancer is unclear. Over 70 years ago, the concept of tumour promoting agents driving latent clones to expand was first proposed. In support of this model, recent evidence suggests that human tissue contains a patchwork of mutant clones, some of which harbour oncogenic mutations, and many environmental carcinogens lack a clear mutational signature. We hypothesised that the environmental carcinogen

Mitochondrial 10-formyltetrahydrofolate (10-formyl-THF) is utilized by three mitochondrial enzymes to produce formate for nucleotide synthesis, NADPH for antioxidant defense, and formyl-methionine (fMet) to initiate mitochondrial mRNA translation. One of these enzymes-aldehyde dehydrogenase 1 family member 2 (ALDH1L2)-produces NADPH by catabolizing 10-formyl-THF into CO2 and THF. Using breast cancer cell lines, we show that reduction of ALDH1L2 expression increases ROS levels and the production of both formate and fMet. Both depletion of ALDH1L2 and direct exposure to formate result in enhanced cancer cell migration that is dependent on the expression of the formyl-peptide receptor (FPR). In various tumor models, increased ALDH1L2 expression lowers formate and fMet accumulation and limits metastatic capacity, while human breast cancer samples show a consistent reduction of ALDH1L2 expression in metastases. Together, our data suggest that loss of ALDH1L2 can support metastatic progression by promoting formate and fMet production, resulting in enhanced FPR-dependent signaling.

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low 2-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs and toxicity of therapy toward healthy hematopoietic cells. We studied the role of cyclin-dependent kinase regulatory subunit 1 (CKS1)–dependent protein degradation in primary human AML and healthy hematopoiesis xenograft models in vivo. Using a small-molecule inhibitor (CKS1i), we demonstrate a dual role for CKS1-dependent protein degradation in reducing patient-derived AML blasts in vivo and, importantly, depleting LSCs, whereas inhibition of CKS1 has the opposite effect on normal hematopoiesis, protecting normal hematopoietic stem cells from chemotherapeutic toxicity. Proteomic analysis of responses to CKS1i in our patient-derived xenograft mouse model demonstrate that inhibition of CKS1 in AML leads to hyperactivation of RAC1 and accumulation of lethal reactive oxygen species, whereas healthy hematopoietic cells enter quiescence in response to CKS1i, protecting hematopoietic stem cells. Together, these findings demonstrate that CKS1-dependent proteostasis is a key vulnerability in malignant stem cell biology.

Summary Loss of p53 function contributes to the development of many cancers. While cell-autonomous consequences of p53 mutation have been studied extensively, the role of p53 in regulating the anti-tumor immune response is still poorly understood. Here, we show that loss of p53 in cancer cells modulates the tumor-immune landscape to circumvent immune destruction. Deletion of p53 promotes the recruitment and instruction of suppressive myeloid CD11b+ cells, in part through increased expression of CXCR3/CCR2-associated chemokines and macrophage colony-stimulating factor (M-CSF), and attenuates the CD4+ T helper 1 (Th1) and CD8+ T cell responses in vivo. p53-null tumors also show an accumulation of suppressive regulatory T (Treg) cells. Finally, we show that two key drivers of tumorigenesis, activation of KRAS and deletion of p53, cooperate to promote immune tolerance., Graphical Abstract, Highlights • Tumor-specific loss of p53 delays tumor rejection in immune-competent hosts • p53 loss increases myeloid infiltration through enhanced cytokine secretion • The increase in Treg cells in response to loss of p53 is independent of Kras mutation • Kras mutations coordinate with p53 loss to regulate myeloid recruitment, TP53 is one of the most frequently mutated genes in cancer; however, its significance in controlling tumor-immune crosstalk is not fully understood. Blagih et al. highlight a key role for tumor-associated loss of p53, a common oncogenic event, in regulating myeloid and T cell responses.

Introductory paragraphThe clinical success of targeted cancer therapy is limited by drug resistance that renders cancers lethal in patients1-4. Human tumours can evolve therapy resistance by acquiringde novogenetic alterations and increased heterogeneity via mechanisms that remain incompletely understood1. Here, through parallel analysis of human clinical samples, tumour xenograft and cell line models and murine model systems, we uncover an unanticipated mechanism of therapy-induced adaptation that fuels the evolution of drug resistance. Targeted therapy directed against EGFR and ALK oncoproteins in lung cancer induced adaptations favoring apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC)-mediated genome mutagenesis. In human oncogenicEGFR-driven andALK-driven lung cancers and preclinical models, EGFR or ALK inhibitor treatment induced the expression and DNA mutagenic activity ofAPOBEC3Bvia therapy-mediated activation of NF-κB signaling. Moreover, targeted therapy also mediated downregulation of certain DNA repair enzymes such as UNG2, which normally counteracts APOBEC-catalyzed DNA deamination events. In mutantEGFR-driven lung cancer mouse models, APOBEC3B was detrimental to tumour initiation and yet advantageous to tumour progression during EGFR targeted therapy, consistent with TRACERx data demonstrating subclonal enrichment of APOBEC-mediated mutagenesis. This study reveals how cancers adapt and drive genetic diversity in response to targeted therapy and identifies APOBEC deaminases as future targets for eliciting more durable clinical benefit to targeted cancer therapy.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes(1,2). The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution(1,3,4). Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such asBCL9, MCL1,ARNT(also known asHIF1B),TERTandMYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompassesBCL9, MCL1andARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassingMYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassingCCND1) in HER2(+)breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.Chromosomal instability enables the continuous selection of somatic copy number alterations, which are established as ordered events that often occur in parallel, throughout tumour evolution and metastasis.

Continuous cancer growth is driven by subsets of self-renewing malignant cells. Targeting of uncontrolled self-renewal through inhibition of stem cell-related signaling pathways has proven challenging. Here, we show that cancer cells can be selectively deprived of self-renewal ability by interfering with their epigenetic state. Re-expression of histone H1.0, a tumor-suppressive factor that inhibits cancer cell self-renewal in many cancer types, can be broadly induced by the clinically well-tolerated compound Quisinostat. Through H1.0, Quisinostat inhibits cancer cell self-renewal and halts tumor maintenance without affecting normal stem cell function. Quisinostat also hinders expansion of cells surviving targeted therapy, independently of the cancer types and the resistance mechanism, and inhibits disease relapse in mouse models of lung cancer. Our results identify H1.0 as a major mediator of Quisinostat’s antitumor effect and suggest that sequential administration of targeted therapy and Quisinostat may be a broadly applicable strategy to induce a prolonged response in patients., Targeting self-renewing cancer cells without deleterious side effects in normal tissues has proven challenging. Here, the authors show that the well-tolerated HDAC inhibitor Quisinostat safely inhibits tumor maintenance and disease relapse by restoring high levels of histone H1.0.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non–small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. Significance: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution. This article is highlighted in the In This Issue feature, p. 2355

Lung cancer is the leading cause of cancer-related deaths worldwide. EGFR mutations, which account for about 25% of lung cancer cases, are more commonly associated with lung cancer in never smokers. Although response rates to EGFR tyrosine kinase inhibitors (TKI) used as a first line therapy are high, acquired resistance inevitably occurs. Elevated tumor mutation burden in EGFR mutant lung cancer is associated with a shorter progression free survival, and worse overall survival with both first and second generation TKIs. Increasing our understanding of drivers of mutagenesis in lung cancer is critical in our efforts to prevent tumor reoccurrence and resistance. APOBEC-driven mutagenesis has been shown to occur in multiple cancer types including lung adenocarcinoma. Using the TCGA and TRACERx datasets we have demonstrated that APOBEC3B is significantly upregulated when compared with other APOBEC family members in EGFR mutant lung adenocarcinoma, and that the APOBEC mutation signature is enriched in a subset of patients with EGFR mutant lung adenocarcinoma. To model APOBEC mutagenesis in lung cancer, we have developed a novel lung cancer mouse model, by crossing the ROSA26LSL-APOBEC3B (A3B) mouse model with several different EGFR mutant mouse models. Using these models, we find that APOBEC3B expression is selected for post-TKI and contributes to resistance to TKI therapy. Citation Format: Deborah Caswell, Manasi Mayekar, Emily Law, William Hill, Eva Gronroos, Reuben Harris, Trever Bivona, Charles Swanton. APOBEC3B and TKI resistance in EGFR mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2938.

BACKGROUND: Aneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events. METHOD: We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset. RESULTS: Cyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors. CONCLUSION: Our study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression.

Background: Cancer evolution occurs though a Darwinian process of natural selection. Selective sweeps occur in most human tumours which obscure early, unsuccessful lineages. In addition, analysis of bulk samples has limited phylogenies to SNVs and Indels, due to methodological difficulties in bulk clonal deconvolution of copy number aberrations (CNAs). Using single cell whole genome sequencing we construct phylogenies of CNAs for murine models of the early stages in lung adenocarcinoma evolution and define features of unsuccessful lineages not yet obscured by full selective sweeps. Methods: Non-invasive tumour tissue was homogenised from EGFRL858R and EGFRL858R/TP53 knock out (KO) mice and subject to 0.1X single cell WGS. Bowtie and AnneuFinder were used to align FASTQ files and call CNAs respectively. A strict quality control step was step applied using read depth and coverage variability, removing 15% of cells. MEDDIC was used to construct phylogenetic trees and ancestral CN states at each node. These ancestral CN states were translated into distinct CN events in the evolutionary history of each tumour. Results: We reconstructed tumour CNA phylogenies using scWGS to determine phylogenetic relationships and time CN events. CNAs in these data were far more heterogenous than in previously published human studies. We noted small populations of progenitor cells which branched off early in evolutionary time, containing far fewer copy number aberrations and which were not genome doubled. These populations allow us to more accurately time CN events such as would be obscured after a full selective sweep. Whole chromosomal events were significantly more likely to occur earlier in evolutionary history than intra-chromosomal events (Wilcoxon test, P < 1e-16, OR = 2.24) and chromosomal gains were also significantly more common at the earlier stages of tumour evolution (Wilcoxon test, P < 1e-16, OR = 1.44). Within whole chromosomal events, gains were significantly more enriched for early timing (Wilcoxon test, P < 1e-16, OR = 7.11). Progenitor cell populations also contained a small number of additional events, which were usually unique to a single cell. We reasoned these events may represent evolutionary dead ends. We found these events were highly enriched for losses in TP53 wild type tumours (Wilcoxon test, P < 1e-16, OR = 11.9) but much less so in TP53 mutant tumours (Wilcoxon test, P = 1e-6, OR = 1.14) suggesting early negative selection against loss of heterozygosity, particularly when TP53 is intact. Discussion: The observed depletion of intrachromosomal events may be caused by a more intact DNA damage response system which is therefore less permissive to chromosomal breaks. Heterozygous losses may be negatively selected due to resultant decreased dosage of a large numbers of genes, upon which the cells become less reliant as cancer progresses. These data are consistent with a recent report of spontaneous CNAs in culture of normal hTERT lines which have a strong preference for gains in a TP53 WT context but not upon TP53 KO. Citation Format: Alexander M. Frankell, Sebastian Hobor, Maise Al-bakir, Eva Grönroos, Charles Swanton. Single cell whole genome sequencing reveals the dynamics of copy number instability at the earliest stages of cancer evolution [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-018.

The DNA replication machinery invariably encounters obstacles that slow replication fork progression, and threaten to prevent complete replication and faithful segregation of sister chromatids. The resulting replication stress activates ATR, the major kinase involved in resolving impaired DNA replication. In addition, replication stress also activates the related kinase ATM, which is required to prevent mitotic segregation errors. However, the molecular mechanism of ATM activation by replication stress is not defined. Here, we show that monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA), a marker of stalled replication forks, interacts with the ATM cofactor ATMIN via WRN-interacting protein 1 (WRNIP1). ATMIN, WRNIP1 and RAD18, the E3 ligase responsible for PCNA monoubiquitination, are specifically required for ATM signalling and 53BP1 focus formation induced by replication stress, not ionising radiation. Thus, WRNIP1 connects PCNA monoubiquitination with ATMIN/ATM to activate ATM signalling in response to replication stress and contribute to the maintenance of genomic stability.

Chromosomal instability (CIN) is the result of ongoing changes in the number (aneuploidy) and structure of chromosomes. CIN is induced by chromosome missegregation in mitosis and leads to karyotypic diversity within the cancer cell population, thereby adding to intratumor heterogeneity. Regardless of the overall pro-oncogenic function of CIN, its onset is typically detrimental for cell fitness and thus tumors must develop CIN-tolerance mechanisms in order to propagate. There is overwhelming genetic and functional evidence linking mutations in the tumor suppressor TP53 with CIN-tolerance. However, the pathways leading to p53 activation following chromosome missegregation remain controversial. Recently, additional mechanisms have been identified in CIN-surveillance, resulting in a more complex network of pathways acting independently or in cooperation with p53. Tolerance might also be achieved by modifying aspects of the cancer cell physiology in order to attenuate CIN or by adaptation to the consequences of aneuploid karyotypes. In this review, we summarize the current knowledge about p53-dependent and -independent mechanisms of CIN-tolerance in cancer, the adaptations observed in CIN cells buffering CIN levels, its consequences for cellular homeostasis, and the potential of exploiting these adaptations in order to design new cancer therapies.

The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with > 10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance. We thank Aida Murra, Naheed Shaikh, Justine Korteweg, Jeremy Tai, Eleanor Carlyle, Leonora Conneely, KimEdmonds, Karla Lingard, Karen O'Meara, Helen Breeze, Sarah Sarker, Lesley Cooper, Linda Shephard, Susie Slater, and Catherine Rogers for study support. We thank the patients and their families. S.T. and H.X. are funded by Cancer Research UK (CRUK) (C50947/A18176). S.T., T.C., J.L., and M.G. are funded by the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) at the Royal Marsden Hospital and Institute of Cancer Research (A109). J.I.L. is funded by the Ministerio de Economia y Competitividad (MINECO, SAF2016-79847-R). M.S., A.S., J. Lynch, R.F., L.A., and L.S. are funded by the Royal Marsden Cancer Charity. K.L. is funded by UK Medical Research Council (MR/P014712/1). T.B.K.W. is funded by the European Union Seventh Framework Programme (FP7-People-2013-ITN). M.J.-H. is funded by the NIHR. N.M.L. is a Winton Group Leader in recognition of the Winton Charitable Foundation's support towards the establishment of The Francis Crick Institute. N.M.L. is additionally funded by a Wellcome Trust Joint Investigator Award (103760/Z/14/Z) and the MRC eMedLab Medical Bioinformatics Infrastructure Award (MR/L016311/1). M.K. is funded by the Danish Cancer Society grant (R90-A6213). P.C. is funded by the Wellcome Trust (WT088340MA). N.M. receives funding from CRUK, Rosetrees, and the NIHR BRC at University College London Hospitals. C.S. is a Royal Society Napier Research Professor. C.S. is funded by Cancer Research UK (TRACERx and CRUK Cancer Immunotherapy Catalyst Network), the CRUK Lung Cancer Centre of Excellence, Stand Up 2 Cancer (SU2C), the Rosetrees and Stoneygate Trusts, NovoNordisk Foundation (THESEUS), Marie Curie Network PloidyNet, the NIHR BRC at University College London Hospitals, and the CRUK University College London Experimental Cancer Medicine Centre. C.S., O.P., D.R., I.C., and Z.S. are funded by NovoNordisk Foundation (16584). O.P., D.R., and I.C. are funded by the National Research, Development and Innovation Office of Hungary (NVKP_16-1-20160004). This work was supported by CRUK (Clinical Scientist Fellowship to S.T., C50947/A18176), The Francis Crick Institute, which receives its core funding from Cancer Reseach UK (FC010110), the UK Medical Research Council (FC010110), the Wellcome Trust (FC010110), and NIHR BRC at the Royal Marsden Hospital and Institute of Cancer Research (A109). High-performance computing was supported by eMedLab. The results published here are in whole or part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.

Summary Chromosomal instability (CIN) contributes to cancer evolution, intratumor heterogeneity, and drug resistance. CIN is driven by chromosome segregation errors and a tolerance phenotype that permits the propagation of aneuploid genomes. Through genomic analysis of colorectal cancers and cell lines, we find frequent loss of heterozygosity and mutations in BCL9L in aneuploid tumors. BCL9L deficiency promoted tolerance of chromosome missegregation events, propagation of aneuploidy, and genetic heterogeneity in xenograft models likely through modulation of Wnt signaling. We find that BCL9L dysfunction contributes to aneuploidy tolerance in both TP53-WT and mutant cells by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID. Efforts to exploit aneuploidy tolerance mechanisms and the BCL9L/caspase-2/BID axis may limit cancer diversity and evolution., Graphical Abstract, Highlights • Loss-of-function alterations in BCL9L are frequent in aneuploid CRC • BCL9L dysfunction drives aneuploidy tolerance by reducing levels of caspase-2 • Caspase-2 activation following aneuploidy results in MDM2 and BID cleavage • p53 stabilization after chromosome missegregation is regulated by caspase-2, López-García et al. find that BCL9L is often genetically inactivated in human colorectal cancers with chromosomal instability. BCL9L dysfunction promotes aneuploidy tolerance by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID independent of TP53 mutation status.

The contribution of whole-genome doubling to chromosomal instability (CIN) and tumor evolution is unclear. We use long-term culture of isogenic tetraploid cells from a stable diploid colon cancer progenitor to investigate how a genome-doubling event affects genome stability over time. Rare cells that survive genome doubling demonstrate increased tolerance to chromosome aberrations. Tetraploid cells do not exhibit increased frequencies of structural or numerical CIN per chromosome. However, the tolerant phenotype in tetraploid cells, coupled with a doubling of chromosome aberrations per cell, allows chromosome abnormalities to evolve specifically in tetraploids, recapitulating chromosomal changes in genomically complex colorectal tumors. Finally, a genome-doubling event is independently predictive of poor relapse-free survival in early-stage disease in two independent cohorts in multivariate analyses [discovery data: hazard ratio (HR), 4.70, 95% confidence interval (CI), 1.04–21.37; validation data: HR, 1.59, 95% CI, 1.05–2.42]. These data highlight an important role for the tolerance of genome doubling in driving cancer genome evolution. Significance: Our work sheds light on the importance of whole-genome–doubling events in colorectal cancer evolution. We show that tetraploid cells undergo rapid genomic changes and recapitulate the genetic alterations seen in chromosomally unstable tumors. Furthermore, we demonstrate that a genome-doubling event is prognostic of poor relapse-free survival in this disease type. Cancer Discov; 4(2); 175–85. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 131

Summary RAS proteins directly activate PI3-kinases. Mice bearing a germline mutation in the RAS binding domain of the p110α subunit of PI3-kinse are resistant to the development of RAS-driven tumors. However, it is unknown whether interaction of RAS with PI3-kinase is required in established tumors. The need for RAS interaction with p110α in the maintenance of mutant Kras-driven lung tumors was explored using an inducible mouse model. In established tumors, removal of the ability of p110α to interact with RAS causes long-term tumor stasis and partial regression. This is a tumor cell-autonomous effect, which is improved significantly by combination with MEK inhibition. Total removal of p110α expression or activity has comparable effects, albeit with greater toxicities., Highlights • Inducible disruption of PI3-kinase p110α interaction with RAS was modeled in mice • Blocking RAS/p110α interaction in established lung tumors causes partial regression • Blocking RAS/p110α binding has effects similar to p110α deletion on tumor regression • Coordinate MEK inhibition is required for major regression of Kras mutant lung cancer

In vivo cells receive simultaneous signals from multiple extracellular ligands and must integrate and interpret them to respond appropriately. Here we investigate the interplay between pathways downstream of two transforming growth factor β (TGF-β) superfamily members, bone morphogenetic protein (BMP) and TGF-β. We show that in multiple cell lines, TGF-β potently inhibits BMP-induced transcription at the level of both BMP-responsive reporter genes and endogenous BMP target genes. This inhibitory effect requires the TGF-β type I receptor ALK5 and is independent of new protein synthesis. Strikingly, we show that Smad3 is required for TGF-β's inhibitory effects, whereas Smad2 is not. We go on to demonstrate that TGF-β induces the formation of complexes comprising phosphorylated Smad1/5 and Smad3, which bind to BMP-responsive elements in vitro and in vivo and mediate TGF-β-induced transcriptional repression. Furthermore, loss of Smad3 confers on TGF-β the ability to induce transcription via BMP-responsive elements. Our results therefore suggest that not only is Smad3 important for mediating TGF-β's inhibitory effects on BMP signaling but it also plays a critical role in restricting the transcriptional output in response to TGF-β.

Background Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. Methods To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. Results Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. Conclusions Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.)

Nature 545, 446–451 (2017); doi:10.1038/nature22364 For 6 of the 96 patients included in this Article (patients CRUK0014, CRUK0030, CRUK0048, CRUK0059, CRUK0096 and CRUK0097) incorrect tumour volumetric data and positron emission tomography (PET) tumour background ratio (TBR) data were analysed. This error occurred because of the incorrect assignment of patient identifiers during the anonymization mandated by the independent review board of pre-operative computed tomography (CT) scans belonging to these patients.

Background: Chromosomal instability (CIN) has been shown to be associated with drug resistance and poor clinical outcome in several cancer types. However, in oestrogen receptor (ER)-negative breast cancer we have previously demonstrated that extreme CIN is associated with improved clinical outcome, consistent with a negative impact of CIN on tumour fitness and growth. The aim of this current study was to validate this finding using previously defined CIN thresholds in a much larger prospective cohort from a randomised, controlled, clinical trial. Patients and methods: As a surrogate measurement of CIN, dual centromeric fluorescence in situ hybridisation was performed for both chromosomes 2 and 15 on 1173 tumours from the breast cancer TACT trial (CRUK01/ 001). Each tumour was scored manually and the mean percentage of cells deviating from the modal centromere number was used to define four CIN groups (MCD1-4), where tumours in the MCD4 group were defined as having extreme CIN. Results: In a multivariate analysis of disease-free survival, with a median follow-up of 91 months, increasing CIN was associated with improved outcome in patients with ER-negative cancer (P trend = 0.03). A similar pattern was seen in ERnegative/ HER2-negative cancers (Ptrend = 0.007). Conclusions: This prospective validation cohort study further substantiated the association between extreme CIN and improved outcome in ER-negative breast cancers. Identifying such patients with extreme CIN may help distinguish good from poor prognostic groups, and therefore support treatment and risk stratification in this aggressive breast cancer subtype.

Phosphorylation-dependent activation of the transcription factors Smad2 and Smad3 plays an important role in TGFbeta-dependent signal transduction. Following phosphorylation of Smad2 and Smad3, these molecules are translocated to the nucleus where they interact with coactivators and/or corepressors, including p300, CBP, and P/CAF, and regulate the expression of TGFbeta target genes. In the current study, we demonstrate that both Smad2 and Smad3 are acetylated by the coactivators p300 and CBP in a TGFbeta-dependent manner. Smad2 is also acetylated by P/CAF. The acetylation of Smad2 was significantly higher than that of Smad3. Lys(19) in the MH1 domain was identified as the major acetylated residue in both the long and short isoform of Smad2. Mutation of Lys(19) also reduced the p300-mediated acetylation of Smad3. By generating acetyl-Lys(19)-specific antibodies, we demonstrate that endogenous Smad2 is acetylated on this residue in response to TGFbeta signaling. Acetylation of the short isoform of Smad2 improves its DNA binding activity in vitro and enhances its association with target promoters in vivo, thereby augmenting its transcriptional activity. Acetylation of Lys(19) also enhanced the DNA binding activity of Smad3. Our data indicate that acetylation of Lys(19) induces a conformational change in the MH1 domain of the short isoform of Smad2, thereby making its DNA binding domain accessible for interactions with DNA. Thus, coactivator-mediated acetylation of receptor-activated Smad molecules could represent a novel way to regulate TGFbeta signaling.

Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

Transforming growth factor beta (TGFbeta) regulates multiple cellular processes via activation of Smad signaling pathways. We have recently demonstrated that the inhibitory Smad7 interacts with the acetyl transferase p300 and that p300 acetylates Smad7 on two lysine residues. These lysine residues are critical for Smurf-mediated ubiquitination of Smad7, and acetylation protects Smad7 from TGFbeta-induced degradation. In this study we demonstrate that Smad7 interacts with specific histone deacetylases (HDACs) and that the same HDACs are able to deacetylate Smad7. The interaction with HDACs is dependent on the C-terminal MH2 domain of Smad7. In addition, HDAC1-mediated deacetylation of Smad7 decreases the stability of Smad7 by enhancing its ubiquitination. Thus, our results demonstrate that the degradation of Smad7 is regulated by the balance between acetylation, deacetylation and ubiquitination, indicating that this could be a general mechanism to regulate the stability of cellular proteins.