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Assessing APOBEC3 gene expression using immunohistochemical and bioinformatic approaches.

APOBEC3 gene expression during senescence and cell cycle progression.

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.

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.

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

Proteolysis-targeting chimeras (PROTACs) are an emerging drug modality that may offer new opportunities to circumvent some of the limitations associated with traditional small-molecule therapeutics. By analogy with the concept of the 'druggable genome', the question arises as to which potential drug targets might PROTAC-mediated protein degradation be most applicable. Here, we present a systematic approach to the assessment of the PROTAC tractability (PROTACtability) of protein targets using a series of criteria based on data and information from a diverse range of relevant publicly available resources. Our approach could support decision-making on whether or not a particular target may be amenable to modulation using a PROTAC. Using our approach, we identified 1,067 proteins of the human proteome that have not yet been described in the literature as PROTAC targets that offer potential opportunities for future PROTAC-based efforts.

Chromosomal instability (CIN), the increase in the rate of whole/partial chromosome gains and losses, is a driving feature of cancer identified in ~90% of solid tumours. CIN generates intratumor heterogeneity (ITH), drives evolutionary adaptation, and is associated with highly aggressive, drug-resistant tumours and poor prognosis. Aberrations in numerous pathways have been suggested to increase CIN. However, how CIN is initiated and maintained in non-small cell lung cancer (NSCLC) is largely unknown. Here we have analysed multi-region WES sequencing data from patients of the TRACERx study, and identified novel genes involved in the DNA damage response (DDR) that may contribute to chromosomal instability. Using orthogonal methods, we have identified that genetic alteration in 6 genes, particularly FAT1, culminates in deficiencies in homologous recombination repair (HRR). This is manifested by a reduction of RAD51 and BRCA1 foci formation, reduced end-resection rate and increased 53BP1 bodies. We find that these deficiencies in the HRR pathway can lead to an increase in structural CIN and deviation from the modal cjromosome number. Furthermore, we observed an early selection of FAT1 mutations in the TRACERx421 LUSC cohort, which resulted in more genome-doubling. We reprot mirrored sub-clonal allelic imbalance (MSAI) events at the FAT1 gene locus, indicating parallel evolution. We have proceeded to validate these observations using several cell lines, showing that FAT1 loss promotes whole genome doubling (WGD) through the generation of actively replicating binucleated cells through an elevated mitotic error rate. Importantly, the elevated CIN induced by FAT1 loss could be partially ameliorated by co-depletion of the oncogene YAP1. We hypothesize that elevated CIN could synergize with WGD to generate increased intratumor heterogeneity. To investigate this, we modelled the relationship between FAT1 loss and genome doubling in an isogenic cell culture system using PC9 cells which are sensitive to the EGFR inhibitor, Osimertinib. Indeed, we observed an elevated rate of acquired resistance in FAT1 KO genome-doubled PC9 cells, which we attribute to the high CIN level generated by FAT1 loss. In line with our hypothesis, we also observed a significant increase in genome content in FAT1 KO cells, suggesting FAT1 loss induces a second WGD event to escape targeted therapy. In conclusion, FAT1 is one of the most frequently mutated genes both in the TRACERx421 cohort and in somatic tissues (Martincorena et al, Science 2015). We postulate that FAT1 loss attenuates DDR and exacerbates CIN to enhance tumour heterogeneity, early in lung cancer evolution. Our observation that FAT1 loss leads to elevated EGFRi resistance also provides a unique opportunity to understand how EGFRi resistance arises through elevated CIN. Citation Format: Wei-Ting Lu, Lykourgos-Panagiotis Zalmas, Chris Bailey, Oriol Pich, Carlos M. Ruiz, James Black, Georgia Stavrou, Dhruva Biswas, Francisco Gimeno-Valiente, Kevin Litchfield, Jiri Bartek, Nicholas McGranahan, Nnennaya Kanu, Charles Swanton. Functional characterisation of TRACERx reveals mechanisms of NSCLC evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB237.

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

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.

Summary Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis., Graphical Abstract, Highlights • A role for SETD2 in DSB resection and homologous recombination repair • Histone H3K36me3 is required for homologous recombination • SETD2 and RAD51 suppress mutations arising from microhomology-mediated end-joining • Mutations affecting H3K36me3 levels may promote tumorigenesis, The SETD2 gene encodes the histone H3K36 trimethyltransferase. Pfister et al. now show that human SETD2-dependent H3K36me3 maintains genome stability by promoting error-free DNA repair through homologous recombination (HR). Upon DNA damage, SETD2-depleted cells exhibit reduced DNA resection, impaired recruitment of early HR factors, and increased utilization of the error-prone microhomology-mediated end-joining repair pathway. Eliminating H3K36me3 by overexpressing the oncogene KDM4A also impairs HR. Thus, H3K36me3 suppresses tumorigenesis by promoting accurate DNA repair.

TRACERx is a longitudinal clinical study, which explores the evolutionary trajectories of NSCLC by temporally dissecting genomic aberrations through multi-region sequencing. Analysis of the first 100 patients revealed extensive intra-tumor heterogeneity (ITH) and chromosomal instability (CIN), manifesting as subclonal somatic mutations and copy number aberrations. Although mutations in “trunk” clonal drivers such as Ras, C-Myc, p53 or MAPK pathway have been thoroughly characterized after the herculean efforts of TCGA consortium, the roles of sub-clonal drivers remain largely obscure as their identification is only made possible by multi-region sequencing. Harnessing the power of the TracerX consortium, we investigate the molecular mechanisms that underpins the role of sub-clonal drivers in ITH. By means of high content imaging of DNA damage markers and tracking human artificial chromosome loss, we aim to elucidate the link between subclonal drivers and CIN events (Zalmas et al, in press). Importantly, we found the ablation of a large proportion of subclonal drivers result in genomic instability. This suggests that genome instability is not merely a standalone hallmarks of cancer, but also a major step that drivers sub-clonal expansion and functionally connect genome instability to tumor evolution. However, the molecular pathways directly linking genome maintenance, chromosomal instability events and cancer evolution is unclear. More intriguingly, only a fraction of these subclonal drivers has been previously reported to be involved in canonical DNA damage response (DDR). This suggests the error-prone DNA repair is favored during tumor clonal evolution. Here, we present previously uncharacterized subclonal driver that are important for error-free DNA repair pathway. The ablation of these genes causes mis-localization of key homologous repair (HR) proteins, such as BRCA1 and Rad51, to DNA damage sites after IR irradiation. Strikingly, early signaling events such as ATM phosphorylation, phosphorylation and ubiquitylation of histone H2A.X, and 53BP1 localization remain unaffected. The exact molecular mechanism the leads to DNA repair pathway selection will be discussed. Citation Format: Wei-ting Lu, Lykourgos-Panagiotis Zalmas, Thomas Webber, Nnennaya Kanu, Charles Swanton. TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2567.

The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity. © 2013 Elsevier Inc.

The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy.

Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.

Background: Loss of the histone mark H3 Lysine 36 trimethylation (H3K36me3) is highly prevalent and is associated with poor prognosis. However, there is currently no therapy targeting H3K36me3-deficient cancers. Recently we found a novel synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to inhibition of WEE1 (a checkpoint kinase). Here we provide our latest and detailed understanding of the underlying mechanism and report our findings in biomarker development. Methods: SETD2 is the sole methyltransferase responsible for generating H3K36me3. We used CRISPR-Cas9 methodology to knockout expression of SETD2 in order to create isogenic cancer cell lines that are negative for H3K36me3. Survival of these isogenic cell lines was measured after treatment with the WEE1 inhibitor AZD1775. Recruitment of transcription factors to the chromatin was analyzed by chromatin immunoprecipitation (ChIP). Rescue experiments were performed by expressing a mutant ribonucleotide reductase (RRM2) that cannot be phosphorylated by CDK (Cyclin-dependent kinase) and is resistant to subsequent degradation by SCF(CyclinF). Immunohistochemistry (IHC) was performed on patient tissue microarrays (>100 samples per cancer type) using a monoclonal antibody against H3K36me3. Results: We showed that SETD2 CRISPR knockout cells are 12-fold more sensitive to the WEE1 inhibitor AZD1775 than parental cells (IC50 = 10 nM vs. 128 nM, p Conclusions: Our study has uncovered the mechanism for the selective killing of H3K36me3-deficient cancers by inhibition of WEE1. We identified novel roles for histone H3K36me3 in promoting gene transcription and DNA replication, and for WEE1 in maintaining nucleotide pools by controlling RRM2 degradation. Our proposed treatment of H3K36me3-deficient cancers is based on synthetic lethality, which provides a less toxic and more effective treatment as it specifically targets cancer cells. Our biomarker analyses revealed that H3K36me3 loss is highly prevalent in renal, colorectal and lung cancers, suggesting that it could be an important therapeutic target in these cancers. H3K36me3 loss can be used as a predictive biomarker for WEE1 inhibitor treatment, and may enable patient selection through immunohistochemistry. Finally, as the WEE1 inhibitor AZD1775, for which we describe a new target, is in clinical trials, we anticipate that these findings will be of immediate clinical relevance. Citation Format: Sophia X. Pfister, Enni Markkanen, Yanyan Jiang, Sovan Sarkar, Mick Woodcock, Lykourgos-Panagiotis Zalmas, Giulia Orlando, Neele Drobnitzky, Grigory Dianov, Songmin Ying, Nicholas B. La Thangue, Vincenzo D'Angiolella, Anderson Ryan, Timothy C. Humphrey. WEE1 inhibition selectively kills histone H3K36me3-deficient cancers by dNTP starvation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B40.

Background The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. Results HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo. HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo. Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro. APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. Conclusion These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1042-9) contains supplementary material, which is available to authorized users.