Studying clonal dynamics in response to cancer therapy using high-complexity barcoding

Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.

[1]  Tony Z. Jia,et al.  Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes , 2012, Proceedings of the National Academy of Sciences.

[2]  S. Kobayashi,et al.  Serum concentrations of Erlotinib at a dose of 25 mg daily. , 2010, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[3]  Qun Zhong,et al.  Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR. , 2011, Lab on a chip.

[4]  K. Kinzler,et al.  Evaluation of DNA from the Papanicolaou Test to Detect Ovarian and Endometrial Cancers , 2013, Science Translational Medicine.

[5]  Jesse J. Salk,et al.  Detection of ultra-rare mutations by next-generation sequencing , 2012, Proceedings of the National Academy of Sciences.

[6]  Johannes G. Reiter,et al.  The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers , 2012, Nature.

[7]  William Pao,et al.  Epidermal growth factor receptor tyrosine kinase inhibitor-resistant disease. , 2013, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[8]  Joon-Oh Park,et al.  MET Amplification Leads to Gefitinib Resistance in Lung Cancer by Activating ERBB3 Signaling , 2007, Science.

[9]  Chuanfeng Wu,et al.  Clonal tracking of rhesus macaque hematopoiesis highlights a distinct lineage origin for natural killer cells. , 2014, Cell stem cell.

[10]  Benjamin E. Gross,et al.  Integrative Analysis of Complex Cancer Genomics and Clinical Profiles Using the cBioPortal , 2013, Science Signaling.

[11]  T. Kunkel.  DNA Replication Fidelity* , 2004, Journal of Biological Chemistry.

[12]  K. Kinzler,et al.  Detection and quantification of rare mutations with massively parallel sequencing , 2011, Proceedings of the National Academy of Sciences.

[13]  Joshua F. McMichael,et al.  Clonal evolution in relapsed acute myeloid leukemia revealed by whole genome sequencing , 2011, Nature.

[14]  P. A. Futreal,et al.  Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. , 2012, The New England journal of medicine.

[15]  S. Gabriel,et al.  EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy , 2004, Science.

[16]  John T. Powers,et al.  Targeting Bcr–Abl by combining allosteric with ATP-binding-site inhibitors , 2010, Nature.

[17]  J. Mestan,et al.  Allosteric inhibitors of Bcr-abl–dependent cell proliferation , 2006, Nature chemical biology.

[18]  K. Pienta,et al.  Copy number and targeted mutational analysis reveals novel somatic events in metastatic prostate tumors. , 2011, Genome research.

[19]  Ben S. Wittner,et al.  A Chromatin-Mediated Reversible Drug-Tolerant State in Cancer Cell Subpopulations , 2010, Cell.

[20]  Pengyuan Liu,et al.  Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer , 2014, Proceedings of the National Academy of Sciences.

[21]  K. Robasky,et al.  The role of replicates for error mitigation in next-generation sequencing , 2013, Nature Reviews Genetics.

[22]  Solomon H. Snyder,et al.  Erratum: Inositol polyphosphate multikinase is a coactivator of p53-mediated transcription and cell death (Science Signaling (2013) 6: 273 (er1) Doi: 10.1126/scisignal.6273er1) , 2013 .

[23]  P. Johnston,et al.  Cancer drug resistance: an evolving paradigm , 2013, Nature Reviews Cancer.

[24]  Joshua M. Korn,et al.  An F876L mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 (enzalutamide). , 2013, Cancer discovery.

[25]  Irving L. Weissman,et al.  Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding , 2011, Nature Biotechnology.

[26]  R. Wilson,et al.  EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Patricia L. Harris,et al.  Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. , 2004, The New England journal of medicine.

[28]  Dominik Wodarz,et al.  Drug resistance in cancer: principles of emergence and prevention. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Adam A. Margolin,et al.  The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity , 2012, Nature.

[30]  S. Ramaswamy,et al.  Systematic identification of genomic markers of drug sensitivity in cancer cells , 2012, Nature.

[31]  James D. Griffin,et al.  Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia , 2007, Nature Reviews Cancer.

[32]  K. Frazer,et al.  Microdroplet-based PCR amplification for large scale targeted sequencing , 2009, Nature Biotechnology.

[33]  M. Iacono,et al.  Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain. , 2013, Blood.

[34]  J. D. Benson,et al.  Contribution of Polycomb Homologues Bmi-1 and Mel-18 to Medulloblastoma Pathogenesis , 2007, Molecular and Cellular Biology.

[35]  Frank Diehl,et al.  BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions , 2006, Nature Methods.

[36]  Lior Pachter,et al.  Sequence Analysis , 2020, Definitions.

[37]  Krishnendu Chatterjee,et al.  Evolutionary dynamics of cancer in response to targeted combination therapy , 2013, eLife.

[38]  William Pao,et al.  Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1 , 2012, Proceedings of the National Academy of Sciences.

[39]  Katherine R. Singleton,et al.  A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop , 2013, Oncogenesis.

[40]  William Pao,et al.  MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib , 2007, Proceedings of the National Academy of Sciences.

[41]  Joshua M. Korn,et al.  Pharmacological and genomic profiling identifies NF-κB–targeted treatment strategies for mantle cell lymphoma , 2013, Nature Medicine.

[42]  Simona Soverini,et al.  BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet. , 2011, Blood.

[43]  Enzo Medico,et al.  Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer , 2012, Nature.

[44]  M. DePristo,et al.  A framework for variation discovery and genotyping using next-generation DNA sequencing data , 2011, Nature Genetics.

[45]  Joshua M. Korn,et al.  An F 876 L Mutation in Androgen Receptor Confers Genetic and Phenotypic Resistance to MDV 3100 ( Enzalutamide ) , 2013 .

[46]  Michael Peyton,et al.  Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer. , 2005, Cancer research.

[47]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[48]  Martin C. Müller,et al.  The quantitative level of T315I mutated BCR-ABL predicts for major molecular response to second-line nilotinib or dasatinib treatment in patients with chronic myeloid leukemia , 2013, Haematologica.

[49]  T. Schumacher,et al.  Diverse and heritable lineage imprinting of early haematopoietic progenitors , 2013, Nature.

[50]  Michael Peyton,et al.  An Epithelial–Mesenchymal Transition Gene Signature Predicts Resistance to EGFR and PI3K Inhibitors and Identifies Axl as a Therapeutic Target for Overcoming EGFR Inhibitor Resistance , 2012, Clinical Cancer Research.

[51]  Cole Trapnell,et al.  Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. , 2010, Nature biotechnology.

[52]  I. Miyoshi,et al.  Establishment of a Ph1 chromosome-positive cell line from chronic myelogenous leukemia in blast crisis. , 1983, International journal of cell cloning.

[53]  Luca Toschi,et al.  Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. , 2010, Cancer cell.

[54]  Benjamin E. Gross,et al.  The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. , 2012, Cancer discovery.

[55]  A. McKenna,et al.  Evolution and Impact of Subclonal Mutations in Chronic Lymphocytic Leukemia , 2012, Cell.

[56]  P. N. Rao,et al.  Clinical Resistance to STI-571 Cancer Therapy Caused by BCR-ABL Gene Mutation or Amplification , 2001, Science.

[57]  W. Lam,et al.  Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells. , 2013, Cancer research.

[58]  S. Digumarthy,et al.  Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors , 2011, Science Translational Medicine.

[59]  Martin A Nowak,et al.  Evolutionary dynamics of escape from biomedical intervention , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.