IP Library › Granted Patent US 12,559,789
Granted Patent B2
US 12,559,789 · App. 16/479,427 · Granted Feb 24, 2026

Method of targeting patient-specific oncogenes in extrachromosomal DNA to treat glioblastoma

Inventors: Roel Verhaak (West Hartford, CT); Hoon Kim (West Hartford, CT); Ana Decarvalho (Detroit, MI); Tom Mikkelsen (Detroit, MI)
Assignees: The Jackson Laboratory; Henry Ford Health System
C12Q1/6841A61K31/4545A61K31/53A61K49/0008A61P35/00C12Q1/6886
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,559,789
App. No.
16/479,427
Granted
Feb 24, 2026
Kind
B2
Abstract

Provided are methods of targeting patient-specific oncogenes in extrachromosomal DNA (ecDNA) to treat glioma in a human. The present methods include identifying a drug that targets against an oncogene present in ecDNA of a human suffering from glioma, such as glioblastoma. The identified oncogenes present in ecDNA include MET, MET/CAPZA2, MDM2, CDK4, SOX2, PIK3CA, MECOM, PDGFRA, EGFR, MYCN, MYC, TERT, SMARCA4, RP56, FBXW7, CDK6, CCND2, ERBB2, BRCA1, and BAP1. The present methods include identifying a drug targeted against the ecDNA oncogene, which drug inhibits the function of the identified oncogene, so as to inhibit tumor growth or progression of the glioma in the human. Also provided are PDX mouse models to further identify and/or confirm patient-specific drugs that target the identified oncogene(s) present in ecDNA. Also provided are methods of diagnosing gliomas or recurrent gliomas and methods of screening or monitoring for recurrence of gliomas. Further provided are methods of validating a predicted presence of ecDNA in a brain tumor using fluorescence in situ hybridization (FISH). Also provided are methods of screening drug candidates for a patient by implanting different identified drugs that target an identified oncogene into PDX mouse models.

Claims (28)

1 . A method of treating a primary brain tumor with a drug that targets a subject-specific oncogene present in extrachromosomal DNA (ecDNA) in a subject diagnosed with glioma, comprising:

(a) performing whole genome sequencing on a primary brain tumor specimen collected from the subject,

(b) determining that the ecDNA is present in the primary brain tumor specimen based on the whole genome sequencing;

(c) identifying the presence a fused oncogene specific to the subject in the ecDNA;

(d) identifying a drug that targets the fused oncogene;

(e) dissociating the primary brain tumor to produce dissociated tumor cells and growing the cells in vitro to obtain neurospheres;

(f) dissociating the neurospheres into neurosphere cells and implanting the neurosphere cells after 7 to 18 passages into an immunodeficient mouse to generate a patient-derived xenograft (PDX) mouse, and allowing the primary brain tumor-derived neurosphere cells to grow in the PDX mouse for a predetermined time period;

(g) administering the identified drug from step (d) into the PDX mouse;

(h) determining inhibition of tumor growth in the PDX mouse by the drug, wherein inhibition indicates that the identified drug is effective for treating the primary tumor in the subject; and

(i) administering the identified drug to treat the primary tumor in the subject,

wherein the fused oncogene is CDK4/MDM2, BRCA1/ERBB2, or CCND2/CDK4.

2 . The method of claim 1 , wherein the glioma is a glioblastoma.

3 . The method of claim 2 , wherein the subject is an adult human.

4 . The method of claim 1 , wherein the determining in step (b) is performed by sequence alignment followed by identifying misalignment against a reference genome sequence, wherein the misalignment against a reference genome sequence is indicative of the presence of ecDNA.

5 . The method of claim 4 , wherein the determination in step (b) is confirmed by one or both of visual inspection and performing amplicon analysis.

6 . The method of claim 1 , further comprising, identifying in step (c) the presence of more than one fused oncogene specific to the subject, and identifying for each of the identified subject-specific fused oncogenes a drug as targeting against the subject-specific fused oncogene.

7 . The method of claim 1 , further comprising:

verifying the presence of ecDNA determined in step (b) and the fused oncogene identified in step (c) using fluorescence in situ hybridization (FISH).

8 . A method of screening a drug candidate for treatment of a primary brain tumor in a subject diagnosed with glioma, comprising:

(a) performing whole genome sequencing on the primary brain tumor specimen collected from the subject,

(b) determining a presence of an ecDNA in the primary brain tumor specimen based on the whole genome sequencing;

(c) identifying a presence of a fused oncogene specific to the subject in the ecDNA;

(d) identifying a drug that targets the fused oncogene;

(e) dissociating the primary brain tumor to produce dissociated tumor cells and growing the cells in vitro to obtain neurospheres;

(f) dissociating the neurospheres into neurosphere cells and implanting the neurosphere cells after 7 to 18 passages into an immunodeficient mouse to generate a patient-derived xenograft (PDX) mouse, and allowing the primary brain tumor-derived neurosphere cells to grow in the PDX mouse for a predetermined time period;

(g) administering the identified drug from step (d) into the PDX mouse; and

(h) determining inhibition of the tumor growth in the PDX mouse by the drug, wherein inhibition indicates that the identified drug is effective for treating the primary tumor in the subject,

wherein the fused oncogene is CDK4/MDM2, BRCA1/ERBB2, or CCND2/CDK4.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 24, 2019
From: THE JACKSON LABORATORY
To: DECARVALHO, ANA; MIKKELSEN, TOM
Reel/Frame 050472/0812 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 24, 2019
From: KIM, HOON; VERHAAK, ROEL; DECARVALHO, ANA; MIKKELSEN, TOM
To: THE JACKSON LABORATORY
Reel/Frame 050472/0982 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 24, 2019
From: DECARVALHO, ANA; MIKKELSEN, TOM
To: HENRY FORD HEALTH SYSTEM
Reel/Frame 050473/0120 →
Continuity (3)
Provisional Application 62576681 · Oct 25, 2017
Provisional Application 62448625 · Jan 20, 2017
Related Publication 20190360029A1 · Nov 28, 2019
References Cited (82)
US 20170224692A1 · Hao · 2017 [cited by examiner]
WO 9935292A1 · 1999 [cited by applicant]
Nikolaev et al. Extrachromosomal driver mutations in glioblastoma and low-grade glioma. Nature Communications; 2014; 5: 5690; DOI: 10.1038/ncomms6690: p. 1-17. (Year: 2014). [cited by examiner]
Nathanson et al. Targeted Therapy Resistance Mediated by Dynamic Regulation of Extrachromosomal Mutant EGFR DNA. Science; 2014; 343: 72-76. (Year: 2014). [cited by examiner]
Vogt et al. Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma. Nucleic Acids Research; 2014; vol. 42; No. 21: 13194-13205. (Year: 2014). [cited by examiner]
Nikolaev et al. Extrachromosomal driver mutations in glioblastoma and low-grade glioma. Nature Communications; 2014; 5: 5690: p. 1-7. (Year: 2014). [cited by examiner]
Gupta et al. Editorial: Targeted Therapies for Glioblastoma: A Critical Appraisal. Frontiers in Oncology; 2019; 9: 1-4. (Year: 2019). [cited by examiner]
Nathanson et al. Science; 2014; 343: 72-76, cited in IDS. (Year: 2014). [cited by examiner]
Vogt et al. Nucleic Acids Research; 2014;, vol. 42; No. 21: 13194-13205, cited in IDS. (Year: 2014). [cited by examiner]
Nathanson et al. Science; 2014; 343: 72-76, cited in IDS. Supplemental Information. (Year: 2014). [cited by examiner]
Stathias et al. PLOS ONE: 2014: DOI: 10.1371/journal.pone.0115842: p. 1-14. (Year: 2014). [cited by examiner]
Favero et al. Annals of Oncology;2015; 26: 880-887. (Year: 2015). [cited by examiner]
Favero et al. Annals of Oncology;2015; 26: 880-887. Supplemental Information. (Year: 2015). [cited by examiner]
Bai et al. Trends in Molecular Medicine; 2011, vol. 17, No. 6: p. 301-312. (Year: 2011). [cited by examiner]
Joo et al. Cell Reports; 2013;6: 260-273. (Year: 2013). [cited by examiner]
deCarvalho et al. (bioRxiv preprint doi: https://doi.org/10.1101/081158; this version posted Jun. 27, 2017; 30 pages) (Year: 2017). [cited by examiner]
Joo et al. (Cell Reports 3, 260-273, Jan. 31, 2013) (Year: 2013). [cited by examiner]
Favero (Favero et al. Annals of Oncology;2015; 26: 880-887) (Year: 2015). [cited by examiner]
Hasselbach et al. (Hasselbach et al. J. Vis. Exp. (83), e51088, doi:10.3791/51088 (2014) (Year: 2014). [cited by examiner]
Chi et al. (2012. Journal of Oncology, vol. 30, No. 3, p. e30-e33 (Year: 2012). [cited by examiner]
deCarvalho (bioRxiv preprint doi: https://doi.org/10.1101/081158; this version posted Oct. 14, 2016, pp. 1-23 and supplemental information, pp. 1-11) (Year: 2016). [cited by examiner]
Malaney et al. (Cancer Letters 344 (2014) 1-12) (Year: 2014). [cited by examiner]
Andor, et al., “Pan-cancer analysis of the extent and consequences of intratumor heterogeneity.” Nat Med. Jan. 2016 ; 22(1): 105-113. [cited by applicant]
Annibali, D. et al., “Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis.” Nat Commun 5, 4632(2014). [cited by applicant]
Aparicio, et al., “The implications of clonal genome evolution for cancer medicine.” N Engl J Med 2013; 368:842-851. [cited by applicant]
Bao, Z.S. et al.: “RNA-seq of 272 gliomas revealed a novel, recurrent PTPRZI-MET fusion transcript in secondary glioblastomas.” Genome Res 24, 1765-73 (2014). [cited by applicant]
Berezovsky, A.D. et al., “Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and astrocytic differentiation.” Neoplasia 16, Issue 3, 193-206.e25 (2014). [cited by applicant]
Berlin, K. et al., “Assembling Large Genomes with Single-Molecule Sequencing and Locality Sensitive Hashing.” Nat Biotechnol 33, 623-30 (2015). [cited by applicant]
Brennan, C.W. et al., “The Somatic Genomic Landscape of Glioblastoma.” Cell 155, 462-477, Oct. 10, 2013. [cited by applicant]
Ceccarelli, M. et al. “Molecular Profling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma.” Cell 164, 550-63 (2016). [cited by applicant]
Chi, A.S. et al., “Rapid Radiographic and Clinical Improvement After Treatment of a MET-Amplified Recurrent Glioblastoma With a Mesenchymal-Epithelial Transition Inhibitor.” Journal of Clinical Oncology, vol. 30, Issue … [cited by applicant]
Chiang, C. et al. “SpeedSeq: ultra-fast personal genome analysis and interpretation.” Nat Methods. (2015), 12(10): 966-968. (2015). [cited by applicant]
Cibulskis, K. et al., “Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples.” Nat Biotechnol 31, 213-219 (2013). [cited by applicant]
Conway, T. et al., “Xenome—a tool for classifying reads from xenograft samples.” Bioinformatics vol. 28 ISMB 2012, pp. 172-178 (2012). [cited by applicant]
Decarvalho, et al., “Discordant Inheritance of Chromosomal and Extrachromosomal DNA Elements contributes to Dynamic Disease Evolution in Glioblastoma.” bioRxiv, Oct. 14, 2016 (Oct. 14, 2016), XP002780079, DOI: 10.1101/0… [cited by applicant]
Decarvalho, et al., “Gliosarcoma Stem Cells Undergo Glial and Mesenchymal Differentiation In Vivo.” Stem Cells, 2010;28:181-190. [cited by applicant]
Delcher, A.L. et al., “Alignment of whole genomes.” Nucleic Acids Research, 1999, vol. 27, No. 11. p. 2369-76. [cited by applicant]
Dolecek, et al., “CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2005-2009.” Neuro Oncol 14 Suppl 5, vl-49 (2012). [cited by applicant]
Favero, F. et al., “Sequenza: allele-specific copy No. and mutation profiles from tumor sequencing data.” Ann Oncol 26, 64-70 (2015). [cited by applicant]
Forbes, S.A. et al., “COSMIC: exploring the world's knowledge of somatic mutations in human cancer.” Nucleic Acids Res 43, D805-11 (2015). [cited by applicant]
Gibaud, et al., “Characterization at nucleotide resolution of the omogeneously staining region sites of insertion in two cancer cell lines.” Nucleic Acids Research, vol. 41, No. 17, Jul. 2, 2013 (Jul. 2, 2013), pp. 8210… [cited by applicant]
Graveel, C. et al., “Activating MET mutations produce unique tumor profiles in mice with selective duplication of the mutant allele.” PNAS, vol. 101, No. 49, 17198-17203 (2004). [cited by applicant]
Hasselbach, L.A. et al., “Optimization of High Grade Glioma Cell Culture fom Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry” Journal of Visualized Experiments, Jan. 2014, 83, e510… [cited by applicant]
International Search Report and Written Opinion from corresponding PCT Appl. No. PCT/US18/14588 mailed Feb. 5, 2018. [cited by applicant]
Kim, et al., “Whole-genome and multisector exome sequencing of primary and post-treatment glioblastoma reveals patterns of tumor evolution.” Genome Research, 25:316-327 (2015). [cited by applicant]
Kim, H.P. et al., “Novel fusion transcripts in human gastric cancer revealed by transcriptome analysis.” Oncogene vol. 33, pp. 5434-5441 (2014). [cited by applicant]
Kim, J. et al., “Spatiotemporal Evolution of the Primary Glioblastoma Genome.” Cancer Cell 28, 318-328 (2015). [cited by applicant]
Kohl, N.E. et al., “Transposition and Amplification of Oncogene-Related Sequences in Human Neuroblastomas.” Cell vol. 35, Issue 2, Part 1, Dec. 1983, 359-367. [cited by applicant]
Li, H. et al., “Fast and accurate short read alignment with Burrows-Wheeler transform.” Bioinformatics, vol. 25 No. 14 2009, pp. 1754-1760. [cited by applicant]
Liu, X. et al., “A Novel Kinase Inhibitor, INCB28060, Blocks c-MET-Dependent Signaling, Neoplastic Activities, and Cross-Talk with EGFR and HER-3.” Clin Cancer Res; (2011); 17(22); 7127-38. [cited by applicant]
Lundberg, G. et al., “Binomial Mitotic Segregation of MYCN-Carrying Double Minutes in Neuroblastoma Illustrates the Role of Randomness in Oncogene Amplification.” PLoS One, vol. 3, Issue 8, e3099. (2008). [cited by applicant]
Mckenna, et al. “The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. ”Genome Res 20, 1297-303 (2010). [cited by applicant]
Mueller, H.W. et al., “Identification of an amplified gene cluster in glioma including two novel amplified genes solated by exon trapping.” Hum Genet 101, 190-7 (1997). [cited by applicant]
Nathanson, et al., “Targeted Therapy Resistance Mediated by Dynamic Regulation of Extrachromosomal Mutant EGFR DNA.” Science, vol. 343, No. 6166, Jan. 2014, pp. 72-76. [cited by applicant]
Nikolaev S., et al., “Extrachromosomal driver mutations in glioblastoma and low-grade glioma.” Nat Comm 5, 5690 (2014) p. 1-7. [cited by applicant]
Organ, S.L. et al., “An overview of the c-MET signaling pathway.” Ther. Adv. Med, Oncol. 3, S7-S19 (2011). [cited by applicant]
Ozawa, T. et al., “Most human non-GCIMP glioblastoma subtypes evolve from a cornmon proneural-like precursor glioma.” Cancer Cell 26, 288-300 (2014). [cited by applicant]
Robinson, J.T., et al., “Integrative genomics viewer.” Nat Biotechnol 29, 24-6 (2011). [cited by applicant]
Roos, W.P. et al., “DNA damage and the balance between survival and death in cancer biology.” Nature Reviews, vol. 16, (2016). [cited by applicant]
Roth, A. et al., “PyClone: Statistical inference of clonal population structure in cancer.” Nat Methods. Apr. 2014 ; 11(4): 396-398. [cited by applicant]
Rubio-Perez, C. et al., “In Silico Prescription of Anticancer Drugs to Cohorts of 28 Tumor Types Reveals Targeting Opportunities.” Cancer Cell 27, 382-396, 2015. [cited by applicant]
Sanborn, J.Z. et al., “Double Minute Chromosomes in Glioblastoma Multiforme Are Revealed by Precise Reconstruction of Oncogenic Amplicons.” Cancer Res; 73(19); 6036-45, (2013). [cited by applicant]
Sequist, et al., “Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors.” Sci Transl Med. Mar. 23, 2011; 3(75): 75ra26. [cited by applicant]
Singh, D. et al., “Transforming Fusions of FGFR and TACC Genes in Human Glioblastoma.” Science. Sep. 7, 2012; 337(6099): 1231-1235. [cited by applicant]
Snuderl, M. et al., “Mosaic Amplification of Multiple Receptor Tyrosine Kinase Genes in Glioblastoma.” Cancer Cell 20, 810-817, Dec. 13, 2011. [cited by applicant]
Sottoriva, A. et al., “Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics.” PNAS, vol. 110, No. 10, 4009-4014 (2013). [cited by applicant]
Storlazzi, C.T. et al., Gene amplification as doubleminutes or homogeneously staining regions in solid tumors: Origin and structure, Genome Res 20, 1198-1206 (2010). [cited by applicant]
Szerhp, NJ. et al., “Intratumoral heterogeneity of receptor tyrosine kinases :GFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response.” Proc Natl Acad. Sci. USA 109, 3041… [cited by applicant]
Thomas, et al., “Glioblastoma-related gene mutations and over-expression of functional epidermal growth factor receptors in SKMG-3 glioma cells.” Acta Neuropathologica, vol. 101, 2001, 605-615. [cited by applicant]
Torres-Garcia, et al., “PRADA: pipeline for RNA sequencing data analysis.” Bioinformatics, vol. 30 No. 15 2014, pp. 2224-2226. [cited by applicant]
Turner, K.M. et al., “Extrachromosomal oncogene amplification drives tumor evolution and genetic heterogeneity.” Nature. Mar. 2, 2017; 543(7643): 122-125. [cited by applicant]
Verhaak, R.G. et al. “Integrated Genomic Analysis Identifies Clinically Relevant Subtypes of Glioblastoma Characterized by Abnormalities in PDGFRA, IDH1, EGFR, and NF1.” vol. 17, Issue 1, Jan. 19, 2010, pp. 98-110. [cited by applicant]
Vogt, et al., “Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma.” Nucleic Acids Research, vol. 42, No. 21, Nov. 6, 2014, pp. 13194-13205. [cited by applicant]
Vogt, N et al., “Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas.” Proc Natl Acad Sci USA vol. 101, 11368-73 (2004). [cited by applicant]
Wang, J. et al., “c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells.” PLoS ONE 3(11): e3769 (2008). [cited by applicant]
Wang, K. et al., “ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.” Nucleic Acids Res 38, el64 (2010). [cited by applicant]
Xi, R. et al., “Copy number variation detection in whole-genome sequencing data using the Bayesian information criterion.” E1128-E1136, PNAS, Nov. 15, 2011, vol. 108, No. 46. [cited by applicant]
Yap et al., “Intratumor Heterogeneity: Seeing the Wood for the Trees.” ScienceTranslationalMedicine.org, (2012) vol. 4 Issue 127 127ps10. [cited by applicant]
Ye, K. et al., “Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads.” Bioinformatic, vol. 25 No. 21 2009, pp. 2865-2871. [cited by applicant]
Yoshihara, K., et al., The landscape and therapeutic relevance of cancer-associated transcript fusions. Oncogene 34, 4845-4854 (2015). [cited by applicant]
Zheng, S., et al., “A survey of intragenic breakpoints in glioblastoma identifies a distinct subset associated with poor survival.” Genes & Development 27:1462-1472 (2013). [cited by applicant]
Zou H. et al., “Double minute amplification of mutant PDGF receptor a in a mouse glioma model.” Scientific Reports, vol. 5, No. 1, Feb. 16, 2015. [cited by applicant]