Published OnlineFirst June 16, 2016; DOI: 10.1158/0008-5472.CAN-16-0396 Cancer Therapeutics, Targets, and Chemical Biology Research Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer Daniele Oddo1,2, Erin M. Sennott3, Ludovic Barault1,2, Emanuele Valtorta4, Sabrina Arena1,2, Andrea Cassingena4, Genny Filiciotto1,2, Giulia Marzolla1,2, Elena Elez5, Robin M.J.M. van Geel6, Alice Bartolini2, Giovanni Crisafulli2, Valentina Boscaro7, Jason T. Godfrey3, Michela Buscarino2, Carlotta Cancelliere2, Michael Linnebacher8, Giorgio Corti2, Mauro Truini4, Giulia Siravegna1,2,9, Julieta Grasselli5, Margherita Gallicchio7, Rene Bernards6, Jan H.M. Schellens6, Josep Tabernero5, Jeffrey A. Engelman3,10, Andrea Sartore-Bianchi4, Alberto Bardelli1,2, Salvatore Siena4,11, Ryan B. Corcoran3,10, and Federica Di Nicolantonio1,2 Abstract Although recent clinical trials of BRAF inhibitor combinations BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. have demonstrated improved efficacy in BRAF-mutant colorectal These mechanisms were clinically relevant, as we identified emer- cancer, emergence of acquired resistance limits clinical benefit. gence of a KRAS G12C mutation and increase of mutant BRAF Here, we undertook a comprehensive effort to define mechanisms V600E allele frequency in the circulating tumor DNA of a patient underlying drug resistance with the goal of guiding development at relapse from combined treatment with BRAF and MEK inhibi- of therapeutic strategies to overcome this limitation. We generated tors. To identify therapeutic combinations capable of overcoming a broad panel of BRAF-mutant resistant cell line models across drug resistance, we performed a systematic assessment of candi- seven different clinically relevant drug combinations. Combina- date therapies across the panel of resistant cell lines. Independent torial drug treatments were able to abrogate ERK1/2 phosphor- of the molecular alteration acquired upon drug pressure, most ylation in parental-sensitive cells, but not in their resistant coun- resistant cells retained sensitivity to vertical MAPK pathway sup- terparts, indicating that resistant cells escaped drug treatments pression when combinations of ERK, BRAF, and EGFR inhibitors through one or more mechanisms leading to biochemical reac- were applied. These therapeutic combinations represent promis- tivation of the MAPK signaling pathway. Genotyping of resistant ing strategies for future clinical trials in BRAF-mutant colorectal cells identified gene amplification of EGFR, KRAS, and mutant cancer. Cancer Res; 76(15); 1–12. Ó2016 AACR. Introduction melanomas and 5%–8% of colorectal cancers (1). The most frequent BRAF mutation (V600E) affects the kinase domain, Activating mutations in the BRAF oncogene occur in approx- mimics BRAF phosphorylated state, and leads to constitutive imately 7% of human malignancies, including 50%–60% of activation of the protein (1). In colorectal cancer, BRAF mutations are associated with hypermethylated tumor subtypes and are 1Department of Oncology, University of Torino, Candiolo, Torino, Italy. 2Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy. 3Massa- linked with aggressive, less-differentiated, and therapy-resistant chusetts General Hospital Cancer Center, Boston, Massachusetts. disease (2). Metastatic colorectal cancer (mCRC) patients with 4Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, BRAF – 5 V600E mutant tumors show poor sensitivity to the EGFR- Milan, Italy. Vall d'Hebron University Hospital and Institute of Oncology targeted mAbs panitumumab and cetuximab and display poor (VHIO), Universitat Autonoma de Barcelona, Barcelona, Spain. 6The Netherlands Cancer Institute, Amsterdam, the Netherlands. 7Depart- prognosis with a median overall survival of only about 6 to 9 ment of Drug Science and Technology, University of Turin, Turin, Italy. months (3). 8 Department of General Surgery, Division of Molecular Oncology and BRAF V600E–mutant tumor types do not respond uniformly Immunotherapy, University of Rostock, Rostock, Germany. 9FIRC Insti- tute of Molecular Oncology (IFOM), Milan, Italy. 10Department of Med- to BRAF-targeted therapy (4). Targeted inhibitors of mutant icine, Harvard Medical School, Boston, Massachusetts. 11Department of BRAF alone, or in combination with inhibitors of its down- Oncology, Universita degli Studi di Milano, Milan, Italy. stream effector MEK, induce high response rates in BRAF- Note: Supplementary data for this article are available at Cancer Research mutant melanoma (5, 6); in contrast, a phase I study of mCRC Online (http://cancerres.aacrjournals.org/). patients has shown that the BRAF inhibitor (BRAFi) vemura- Corresponding Author: Federica Di Nicolantonio, Department of Oncology, fenib has no clinical benefit when given as monotherapy (7). University of Torino, Strada Provinciale 142, Km 3.95, Candiolo 10060, Torino, The molecular basis of this discrepancy has been partly Italy. Phone: 3901-1993-3827; Fax: 3901-1993-3225; E-mail: explained by dissimilar EGFR expression levels between these [email protected] two malignancies. Intrinsic resistance of colorectal cancer cells doi: 10.1158/0008-5472.CAN-16-0396 to BRAF or MEK-targeted agents is mediated by the release of Ó2016 American Association for Cancer Research. a feedback loop, which activates EGFR signaling, leading to www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst June 16, 2016; DOI: 10.1158/0008-5472.CAN-16-0396 Oddo et al. reactivation of MAPK signaling and often to upregulation of HROC87 parental cells were shared by M. Linnebacher (Univer- parallel PI3K–AKT pathways, triggering proliferation and sur- sity of Rostock, Rostock, Germany) in September 2011. VACO432 vival (8–10). Melanomas are sensitive to BRAFi as they orig- parental cells were obtained from Horizon Discovery in March inate from the neural crest and do not express EGFR, making 2011. The genetic identity of parental cell lines and their resistant this feedback loop ineffective. On the other hand, colorectal derivatives was confirmed by short tandem repeat profiling (Cell cancers arise from epithelial cells in which EGFR is generally ID System; Promega) not fewer than 2 months before drug constitutively expressed. profiling experiments. BRAF-mutant HROC87, VACO432, These preclinical studies have provided the rationale for testing and WiDr cells were seeded in 100-mm dishes at a density of dual/triple vertical blockade of the MAPK pathway by targeting 5 Â 106/plate and treated with drug combinations as indicated in EGFR, BRAF, and MEK in BRAF-mutant mCRC patients. Combi- Supplementary Table S1. Additional information is provided in nations targeting EGFR, BRAF, and the prosurvival PI3K pathways Supplementary Materials and Methods. are also being explored. Clinical objective responses have been – seen in 20% 40% of patients treated with doublet or triplet Drug sensitivity assay – combinatorial regimens (11 13). Cell proliferation and cytotoxicity were determined by cellular Nevertheless, preliminary clinical evidence from phase Ib trials ATP levels (CellTiter-Glo Luminescent Assay; Promega) and DNA – shows that responses are limited in duration (4, 11 16). The incorporation of a fluorescent cyanine dye (CellTox Green; Pro- molecular basis underlying intrinsic or acquired resistance to mega) after 72-hours drug treatment, respectively. Additional BRAF these drug combinations in -mutant mCRC has not been information is provided in Supplementary Materials and Meth- fi comprehensively de ned. The mechanisms by which cancer cells ods and Supplementary Table S2. evade targeted therapies are usually molecularly heterogeneous, but they often converge downstream in the pathway, which was originally blocked by the targeted agent. For instance, cell lines Western Blot analysis fi and mCRC patients that become resistant to single-agent cetux- Protein quanti cation, SDS-PAGE, Western blotting, and imab or panitumumab show a variety of molecular mechanisms chemiluminescent detection were performed as described previ- that converge in reactivating the MAPK pathway, including muta- ously (19). Detailed information is provided in Supplementary tions in the drug-binding sites of EGFR, RAS/RAF amplification, or Materials and Methods. mutations, or genetic alterations leading to activation of alterna- tive receptor tyrosine kinases (RTK) such as MET or HER2 Gene copy number analysis qPCR (reviewed in ref. 17). Similarly, BRAF-mutant melanomas that Cell line DNA (10 ng) was amplified by quantitative PCR using become refractory to BRAF and/or MEK inhibitors (MEKi) also the GoTaq QPCR Master Mix (Promega) with an ABI PRISM show a variety of molecular mechanisms leading to reactivation of 7900HT apparatus (Applied Biosystems). HER2, MET, EGFR, MAPK and/or AKT signaling. These include increased expression KRAS, and BRAF gene copy number was assessed as described of RTKs such as PDGFRb, IGF-1R, and EGFR; overexpression of the previously (19). Data were normalized to a control diploid cell COT kinase; mutation of MEK1 (MAP2K1) and MEK2 (MAP2K2) line, HCEC (22), and expressed as the ratio between resistant and kinase; MITF or NRAS mutations; amplification, or alternative the corresponding parental cells. Primer sequences are reported in splicing of the BRAF gene; CDKN2A loss; or genetic alterations in Supplementary Table S3. the PI3K–PTEN–AKT pathway