Targetable Fusion Genes in Breast Cancer Rachael Natrajan 1 , Andrew N.J

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IN THE SPOTLIGHT Driver Oncogenes but not as We Know Them: Targetable fusion Genes in Breast Cancer Rachael Natrajan 1 , Andrew N.J. Tutt 1 ,2 , and Christopher J. Lord 1 ,3 summary: Two reports in this issue of Cancer Discovery outline how the genomic composition of tumors, including the presence of intragenic gene fusions, could inform the selection of treatment approaches in aggressive forms of the disease. Cancer Discov; 8(3); 272–5. ©2018 AACR.

See related article by Matissek et al., p. 336 (7). See related article by Liu et al., p. 354 (8).

Despite the implementation of targeted therapy approaches yet distinct form of TNBC, are driven by a t(12;15)(p13;q25) in breast cancer, managing many of the aggressive forms of chromosomal translocation, which encodes a transforming the disease still remains a challenge. For example, despite ETV6–NTRK3 gene fusion ( Fig. 1 ; ref. 2). Likewise, adenoid the improvements in survival that estrogen deprivation ther- cystic carcinomas of the breast are driven by an MYB–NFIB apy has delivered for those with estrogen receptor–positive fusion caused by a t(6;9)(q22–23;p23–24) translocation ( 3 ). (ER-positive) breast cancers, managing endocrine therapy– Other rare, yet recurrent, rearrangements in invasive ductal resistant disease is complex. Likewise, identifying targeted carcinomas of the breast have also been identifi ed, includ- approaches that are effective in those breast cancers that ing those associated with the MAST kinase and NOTCH gene lack estrogen receptor, progesterone receptor, and expression family members ( 1 ). More recently, recurrent gain-of-function of the ERBB2 receptor, the so-called “triple-negative breast fusions involving the gene that encodes the alpha isoform cancers” (TNBC), also remains challenging. One approach to of the estrogen receptor, ESR1 (e.g., ESR1–CCDC170 ), have these issues has been to take a precision medicine approach been identifi ed in aggressive ER-positive, luminal B breast by customizing the treatment strategy based upon the precise cancers; these fusions likely cause constitutive activity of ERα molecular makeup of each individual’s disease. ( 4 ). Next-generation sequencing studies have also identifi ed a Although the delineation of breast cancer genomes and number of breast cancer gene fusions associated with other transcriptomes has heralded an era where this precision medi- well-established cancer driver genes, including BRAF and MET cine approach can be much better implemented, one particu- ( Fig. 1 ; refs. 5, 6 ). lar mutation type, intergenic fusions, that is, chromosomal In this issue of Cancer Discovery, the laboratories of Ellisen (7 ) rearrangements that lead to fusions between two distinct and Cantley (8 ) describe the identifi cation of novel, therapeuti- genes, has received somewhat less attention than might be cally tractable oncogenic fusion genes in ER-positive and triple- expected. This is largely because fusions were historically negative breast cancers, respectively. Importantly, both groups considered to be a feature of leukemias and sarcomas, and to make the critical leap from identifying fusion events to function- have less infl uence upon carcinomas. Furthermore, the tech- ally assessing how these mutations alter therapeutic responses. nical challenges associated with discriminating real fusion In the fi rst report, Matissek and colleagues used AMP to events from false positives are considerable and have limited isolate fusion genes associated with 54 candidate genes in large-scale discovery of intergenic fusions. This scenario has 110 patients with ER-positive breast cancer. This identifi ed changed somewhat, with advances in techniques including intragenic fusions associated with the kinase-coding genes anchored multiplex PCR (AMP), next-generation sequenc- PIK3CA, AKT3, RAF1 , and also ESR1, consistent with prior ing, and sequencing analytic pipelines now making fusion reports ( 1 ). After confi rming some of the fusion events in identifi cation in breast cancers a more profi table exercise both primary and metastatic lesions by an orthogonal tech- ( 1 ). For example, secretory carcinomas of the breast, a rare nology (FISH), Matissek and colleagues used a panel of functional assays to establish that some of the fusions involving the RAF1, PIK3CA, and AKT3 genes modulated phosphopro- 1 The Breast Cancer Now Toby Robins Research Centre, The Institute tein signaling via elevated phosphorylation of the down- of Cancer Research, London, United Kingdom. 2 The Breast Cancer Now stream substrate RPS6 as well as estrogen-dependent growth Research Unit at King’s College Hospital, London, United Kingdom. 3 CRUK in three-dimensional in vitro cultures of breast cancer. Impor- Gene Function Laboratory, The Institute of Cancer Research, London, tantly, the RPS6K1–AKT3 fusion also conferred resistance United Kingdom. to estrogen withdrawal in mice bearing ER-positive tumor Corresponding Authors: Christopher J. Lord, The Institute of Cancer cell line xenografts. An analysis of clinical data suggests that Research, 237 Fulham Road, London SW3 6JB, United Kingdom. Phone: 4420-7153-5190; Fax: 4420-7153-5332; E-mail: [email protected] ; fusions associated with PIK3CA, ESR1, RAF1, or AKT3 are Rachael Natrajan, [email protected] ; and Andrew N.J. Tutt, andrew. relatively uncommon in primary disease but more frequent [email protected] in metastatic ER-positive cancers. Furthermore, those with doi: 10.1158/2159-8290.CD-18-0091 fusion-positive tumors had a shorter overall survival when ©2018 American Association for Cancer Research. compared with those without fusions. In totality, Matissek

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Fusion gene structure Breast cancer subtype Reference

ETV6–NTRK3 Secretory carcinomas (TNBC) Tognon et al. (2) 5-′ SAM Kinase -3′ Chr12p13-Chr15q MYB-NFIB Adenoid–cystic carcinomas (TNBC) Persson et al. (3) 5-′ DBD TADTPTPF UTR -3′ Chr6q23-Chr9p23 various-MAST1/2 Robinson et al. (1) 5-′ Promoter PZD Kinase -3′ ER+/ER− gene x- Chr19q13/Chr1p13 various–NOTCH1/2 ER− Robinson et al. (1) 5-′ Promoter TM NICD -3′ gene x- Chr9q34/Chr1p12 ESR1–CCDC170 + Veeraraghavan et al. (4) 5-′ Promoter SMC Prok B -3′ ER /Luminal B

Chr6q25-Chr6q25 KIAA1549–BRAF Metastatic Ross et al. (5) 5-′ Kinase -3′ Chr7q34-Chr7q34 CAPZA2–MET ER+ Yoshihara et al. (6) 5-′ Kinase -3′

Chr7q31-Chr7q31 FGFR3–TACC3 Shaver et al. 5-′ Kinase Coiled-coil -3′ TNBC Chr7q34-Chr7q34 ACTL6A–PIK3CA ER+ advanced disease Matissek et al. (7) 5-′ Promoter AB RasBC2 Helical Kinase -3′ Chr3q26-Chr3q26 RPS6KC1-AKT3 ER+ advanced disease 5-′ PX Kinase PH -3′ Matissek et al. (7) Chr1q32-Chr7q31 CTNNBL1–RAF1 5-′ Helix Kinase -3′ ER+ advanced disease Matissek et al. (7) Chr20q11-Chr7q31 ESR1–CoA5

5-′ Promoter UTR -3′ ER+ advanced disease Matissek et al. (7) Chr6q25-Chr2q11 Fgfr2–Dnm3 Trp53/Brca1 GEMM Liu et al. (8) 5-′ Ig repeats TM Kinase Coiled-coil -3′ Chr7qF3-Chr1qH2 Dhx9–Raf1 Liu et al. (8) 5-′ DSRM Kinase -3′ Trp53/Brca1 GEMM Chr1qG3-Chr6qE3

Figure 1. Gene fusions in breast cancer. Schematic diagrams of oncogenic gene fusions identified in breast cancers. The breast cancer subtype and functional domains preserved in the gene fusion are noted. 5′ and 3′ untranslated regions (UTR) are depicted by lighter colors. Vertical line, chromosomal breakpoint.

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Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Views and colleagues’ work suggests that oncogenic fusions in ER- effective approach to treating this disease is to take a fully positive breast cancer could function as predictive biomark- personalized approach and target the almost-unique set of ers of clinical resistance to endocrine therapy. molecular aberrations in each patient, rather than seeking One interesting aspect of the work of Matissek and col- therapeutic approaches that might work in many. leagues is the enhanced frequency of fusion events in met- Liu and colleagues test the hypothesis that some of the astatic disease when compared with primary ER-positive relatively private gene mutations that occur in individual cancer; this suggests that fusion genes might, in themselves, TNBCs provide therapeutic vulnerabilities (8). To do this, be a biomarker of advanced and aggressive disease. This asso- Liu and colleagues carried out whole-exome DNA sequenc- ciation with metastatic disease might be expected, given the ing and RNA sequencing from more than 80 TNBCs that enhanced level of genomic alterations in this setting when developed in genetically engineered mice with either a breast compared with primary disease. Furthermore, a number of lineage–specific Trp53 mutation or a combination of Trp53 the fusion events that were private to metastatic lesions, and and Brca1 mutations (8). Similar to previous studies using not found in patient-matched primary lesions, were present the same mouse models (10), the DNA/RNA profiling of prior to any treatment in the metastatic setting. Whether TNBCs by Liu and colleagues identified a recurrent basal-like these are selected for by treatment of primary disease or exist transcriptomic signature and Met oncogene amplification, in clones that have other fitness advantages remains to be and in Trp53/Brca1–mutant mouse tumors an elevated level seen. For many, the therapeutic aspects of this work will be of of genomic rearrangements. Reassuringly, the mutational most interest; although the RPS6K1–AKT3 fusion was associ- spectrum seen in Trp53/Brca1–mutant tumors was most simi- ated with endocrine therapy resistance, the combination of lar to the signature 3 profile associated withBRCA1 or BRCA2 estrogen deprivation with the CDK4/6 inhibitor palbociclib mutations in human tumors. The two most notable discover- caused significant tumor growth suppressionin vivo (7), sug- ies, however, focused upon (i) fusion genes associated with a gesting that looking for this and similar fusions in biopsies series of potentially oncogenic and targetable protein kinases, from clinical trials assessing CDK4/6 inhibitors with endo- such as Fgfr2 or Braf; and (ii) different tumors exhibiting crine therapies might be informative. distinct routes to the same dysregulated pathways, namely Matissek and colleagues’ work is also notable in that, MAPK or PI3K signaling, a potential form of parallel evolu- although most of the literature in the area of fusion genes tion. In some cases, these fusions were shown to be essential in breast cancer has focused upon identifying fusions via for tumor-cell fitness and also therapeutically exploitable; for DNA/RNA sequencing, there is much less emphasis given to example, in mice with Fgfr2 fusions, clinical FGFR inhibitors demonstrating that the fusions identified are oncogenic or impeded tumor growth (8). modulate therapy response. This is perhaps expected, given One interesting aspect of the work of Liu and colleagues the technical challenges faced in experimentally recapitulat- is that tumors with multiple distinct alterations, such as ing chromosomal translocations and the gene fusions they a tumor with both an Fgfr2 fusion and a Brca1 mutation, cause. The expression of cDNAs from expression constructs showed a better response to combination therapies that tar- is normally the approach used to interrogate fusion gene geted both oncogenic drivers (e.g., a FGFR inhibitor plus a function, and this can often be very informative. However, PARP inhibitor) than single-agent approaches that targeted the ectopic pattern of gene expression generated from a only one driver gene. The rationale for such an approach cDNA expression construct often does not replicate that seen might appear at first sight to be self-evident, in that two drugs in tumor cells when the gene is transcribed in its chromo- with distinct mechanisms of action, targeting different driver somal context. Endogenous gene promoters are rarely used effects, are less unlikely to have overlapping mechanisms of with cDNAs, and therefore cDNAs are not transcribed from resistance. However, although much of the field focuses upon within the genomic context of the fusion gene. In models of identifying synergistic drug combinations that could be effec- acute myeloid leukemia and Ewing sarcoma, chromosomal tive as cancer treatments, the approach of simultaneously translocations and the fusion genes they cause have been targeting multiple driver lesions in the same tumor with addi- engineered into cells by CRISPR/Cas9-mediated gene editing tive combinations might be as, or even more, effective. (9), suggesting that this might also be an approach that could Both of these reports highlight the potential of fusion be applied to the study of breast cancer gene fusions. genes as bona fide driver events and determinants of targeted Compared with ER-positive cancers, where targeted endo- therapy responses in breast cancer. The increasing technical crine therapy is widespread but drug resistance is a major advances in sequencing methodologies, especially those that challenge, in TNBC identifying targeted therapies that work aim to increase DNA read lengths, will undoubtedly enhance in significant numbers of patients is the significant issue. the ability to detect these events in the future. Moreover, There are a number of reasons for this; perhaps chief among incorporating fusion gene detection into the analysis of these is the relative absence of unifying and targetable molec- cell-free DNA/circulating tumor cell DNA sequencing will ular features, such as ERα or ERBB2 amplification, in the wide provide the opportunity to monitor these events through the variety of patients with TNBC. Although TNBCs do display course of an individual patient’s clinical journey, information some relatively recurrent molecular alterations (e.g., TP53, RB, that could prove critical in the optimal selection and adjust- NF1, BRCA1, PTEN, or PIK3CA mutations; a series of copy- ment of treatment. number alterations; a basal-like transcriptomic signature, etc.), strategies to target most of these do not exist. Instead, Disclosure of Potential Conflicts of Interest intertumoral molecular heterogeneity is the pervading char- C.J. Lord reports receiving a commercial research grant from acteristic of TNBC; this has led to propositions that the most AstraZeneca and commercial research support from Merck Serono,

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Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. views has received speakers bureau honoraria from AstraZeneca, and is 4. Veeraraghavan J, Tan Y, Cao XX, Kim JA, Wang X, Chamness GC, a consultant/advisory board member AstraZeneca, Sun Pharma, et al. Recurrent ESR1-CCDC170 rearrangements in an aggressive Tango, and Vertex. No potential conflicts of interest were disclosed subset of oestrogen receptor-positive breast cancers. Nat Commun by the other authors. 2014;5:4577. 5. Ross JS, Wang K, Chmielecki J, Gay L, Johnson A, Chudnovsky J, et al. Acknowledgments The distribution of BRAF gene fusions in solid tumors and response to targeted therapy. Int J Cancer 2016;138:881–90. We thank Breast Cancer Now and Cancer Research UK for funding 6. Yoshihara K, Wang Q, Torres-Garcia W, Zheng S, Vegesna R, Kim H, work in our laboratories. et al. The landscape and therapeutic relevance of cancer-associated transcript fusions. Oncogene 2015;34:4845–54. Published online March 2, 2018. 7. Matissek KJ, Onozato ML, Sun S, Zheng Z, Schultz A, Lee J, et al. Expressed gene fusions as frequent drivers of poor outcomes in hormone receptor–positive breast cancer. Cancer Discov 2018;8: 336–53. References 8. Liu H, Murphy CJ, Karreth FA, Emdal KB, Yang K, White FM, . 1 Robinson DR, Kalyana-Sundaram S, Wu YM, Shankar S, Cao X, Ateeq et al. Identifying and targeting sporadic oncogenic genetic aberra- B, et al. Functionally recurrent rearrangements of the MAST kinase tions in mouse models of triple-negative breast cancer. Cancer Discov and Notch gene families in breast cancer. Nat Med 2011;17:1646–51. 2018;8:354–69. 2. Tognon C, Knezevich SR, Huntsman D, Roskelley CD, Melnyk N, 9. Torres R, Martin MC, Garcia A, Cigudosa JC, Ramirez JC, Rodri- Mathers JA, et al. Expression of the ETV6-NTRK3 gene fusion as guez-Perales S. Engineering human tumour-associated chromosomal a primary event in human secretory breast carcinoma. Cancer Cell translocations with the RNA-guided CRISPR-Cas9 system. Nat Com- 2002;2:367–76. mun 2014;5:3964. 3. Persson M, Andren Y, Mark J, Horlings HM, Persson F, Stenman G. 10. Liu X, Holstege H, van der Gulden H, Treur-Mulder M, Zevenhoven Recurrent fusion of MYB and NFIB transcription factor genes in J, Velds A, et al. Somatic loss of BRCA1 and p53 in mice induces carcinomas of the breast and head and neck. Proc Natl Acad Sci U S A mammary tumors with features of human BRCA1-mutated basal-like 2009;106:18740–4. breast cancer. Proc Natl Acad Sci U S A 2007;104:12111–6.

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Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Driver Oncogenes but Not as We Know Them: Targetable Fusion Genes in Breast Cancer

Rachael Natrajan, Andrew N.J. Tutt and Christopher J. Lord

Cancer Discov 2018;8:272-275.

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