HHS Public Access Author manuscript Author Manuscript
Cancer Discov. Author manuscript; available in PMC 2016 December 01. Published in final edited form as: Cancer Discov. 2016 June ; 6(6): 574–575. doi:10.1158/2159-8290.CD-16-0478.
Something Old, Something New, Something Borrowed, Something Fused: Novel EGFR Rearrangements in Lung Adenocarcinomas Paul K. Paik1,2 1Thoracic
Author Manuscript
Oncology Service, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065
2Weill
Cornell Medical College, New York, NY 10065
Summary Mutations in the epidermal growth factor receptor (EGFR) stand as the archetype for somatic alterations that lead to oncogene addiction and that predict for response to targeted therapies. In this issue of Cancer Discovery, Konduri and colleagues report on a pair of novel oncogenic and actionable EGFR fusion events in a series of patients with lung adenocarcinomas, casting new light on this model gene.
Author Manuscript
The shift in classifying non-small cell lung cancers (NSCLC) by their molecular aberrations first and their histologies second, while not yet complete, is a reflection of the growing success of targeted therapeutics in this disease. Where histology once dictated the selection of a matched treatment (i.e. pemetrexed for non-squamous NSCLCs), it is increasingly used now as a guidepost for molecular testing: as a means to a more precise therapeutic end. This has been driven largely by the rapid pace at which actionable oncogenic events have been identified: a driver can now be found in most patients’ tumors. Over the past five years, there has also been a change in the nature of these newly identified targets as access to more comprehensive molecular testing tools has been brought into clinical use.
Author Manuscript
Hence the recent identification of a series of gene rearrangements through next-generation DNA and RNA sequencing (NGS) approaches, including those that involve rearranged during transfection (RET), c-ros oncogene 1 (ROS1), neurotrophic tyrosine kinase receptor 1 (NTRK1), and fibroblast growth factor receptors 1/3 (FGFR1/3).(1–4) These events are rare, not exceeding 1–2% per rearrangement, although they have been immediately translatable given the availability of existing tyrosine kinase inhibitors (TKIs) that have either been FDA-approved or are under investigation in other indications. Commonalities across these rearrangements have been identified, with two features that are generally present. First, each rearrangement contains the intact kinase domain of the relevant protooncogene. Second, the rearrangement partners, while varied, appear to be functionally
Contact information: Paul K. Paik, 300 East 66th Street, New York, NY 10065. 646-888-4202(p); ; Email: [email protected] Conflict of interest: The authors disclose no potential conflicts of interest.
Paik
Page 2
Author Manuscript
important, often containing coiled coil domains which can facilitate dimerization and/or localization of the fusion protein.
Author Manuscript
In this issue of Cancer Discovery, Konduri and colleagues(5) report on a pair of novel fusion events involving the archetype for targeted therapeutics in NSCLC- the epidermal growth factor receptor (EGFR)- and two 3′ partners- RAD51 and PURB- in a series of 5 patients with stage IV lung adenocarcinomas. Each of the patients’ tumors was tested in real-time with the hope of finding an actionable target using a clinically validated hybrid capturebased NGS platform (FoundationOne). Four patients had tumors harboring an EGFR fusion with a 5′ breakpoint within intron 24 (RAD51). The other harbored an EGFR fusion with a breakpoint at exon 25 (PURB). Both, consequently, had retention of an intact EGFR kinase domain. The EGFR variants were fused to either exons 4-9 of RAD51 or the 3′ untranslated region of PURB. Overall, fusion events involving EGFR exons 23-intron 25 proved to be rare, occurring in about 0.05% of more than 10,000 NSCLC tumors tested. Pre-clinical modeling of the EGFR-RAD51 fusion validated its oncogenic potential in vitro. Stable transfection of this variant in Ba/F3 cells led to IL-3 independent growth and downstream activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways to a similar extent as in Ba/F3 cells expressing EGFR L858R. EGFR-RAD51 expression in NR6 cells (an NIH 3T3 variant that lacks endogenous EGFR) was similarly transforming, leading to increased colony formation when compared to parental cells.
Author Manuscript
The authors confirmed that the 1st, 2nd, and 3rd generation EGFR TKIs erlotinib, afatinib, and osimertinib could inhibit growth and signaling in EGFR-RAD51 expressing Ba/F3 cells with low nanomolar IC50s that were not significantly different from those seen in EGFR L858R expressing cells. EGFR-RAD51 expressing cells were, however, more sensitive to growth inhibition by cetuximab than were EGFR L858R expressing cells. These in vitro data were in keeping with the durable partial responses reported in all 4 patients who were treated with erlotinib in this report (N=3 EGFR-RAD51, N=1 EGFR-PURB). The therapeutic potential of this work alone is important for its immediate clinical relevance.
Author Manuscript
As intriguing, however, are the structure-function questions that the fusion events bring to light. RAD51 is a eukaryotic homologous recombinase that self-assembles into filaments containing several RAD51 protomers.(6) Purβ (encoded by PURB) is a member of a family of ssDNA and RNA binding proteins and functions as a transcriptional repressor in vascular smooth muscle cells and myofibroblasts.(7) Prior studies have demonstrated that Purβ can also self-associate into homodimeric complexes.(7) This raises the possibility that both RAD51 and Purβ could facilitate EGFR dimerization, which would be in keeping with the presumed function of the non-kinase partners in other oncogene rearrangements. To test this hypothesis, the authors performed in silico modeling of EGFR-RAD51 based on existing structural data and showed that asymmetric dimerization of EGFR is at least geometrically possible. Whether dimerization does indeed occur in practice in either event is currently unknown.
Cancer Discov. Author manuscript; available in PMC 2016 December 01.
Paik
Page 3
Author Manuscript Author Manuscript
It is worth highlighting in light of this that EGFR C-terminal domain (CTD, spanning exons 25-28) deletions have been previously identified in glioblastomas and lung adenocarcinomas.(8) This domain is effectively deleted in the EGFR-RAD51 and EGFRPURB fusion events. CTD deletions vary in extent, from truncations of the entire CTD to partial deletions of it. At least three of these variants- an exon 27 deletion, exon 25-27 deletion, and exon 25-28 deletion- are transforming, as previously shown by Cho and colleagues.(8) In addition, these CTD variants sensitize cells to growth inhibition with erlotinib or cetuximab. Intriguingly, Cho and colleagues had also identified a novel CTD variant in an EGFR wild-type infected Ba/F3 clone that unexpectedly exhibited IL-3 independent growth. This variant was marked by an intragenic deletion of residues encoding amino acids 1,010 to 1,152 (referred to as CT Del1). Re-expression of the CT Del1 variant into parental Ba/F3 cells and NIH-3T3 cells was transforming. An orthotopic LN443 glioblastoma xenograft model stably expressing CT Del1 also generated brain tumors whose growth could be inhibited by cetuximab.
Author Manuscript
The CT Del1 variant is intriguing because of its focal nature. The deletion includes tyrosine 1045, which serves as the main binding site for the Cbl ubiquitin ligase that targets EGFR for degradation. Consistent with deletion of Y1045, Konduri and colleagues found that the EGFR-RAD51 fusion exhibited decreased EGF receptor turnover. As the authors noted, the CTD also contains a number of auto- and trans-phosphorylated docking sites for adapters that can potentiate EGFR signaling, though these are not necessary for activation of the receptor.(9) Taken together with other experimental data, the work by Konduri and colleagues suggests that the EGFR-RAD51/PURB fusions might increase EGFR activity through two mechanisms- facilitation of asymmetric dimerization and/or impairment of receptor turnover. The latter is the presumptive mechanism of action for the recently identified oncogenic exon 14 deletion in the mesenchymal epithelial transition factor (MET) proto-oncogene, which eliminates the Y1003 Cbl binding site and leads to receptor stabilization, providing a proof-of-principle for this concept.(10) In summary, Konduri and colleagues have identified novel, actionable, and rare EGFR fusions whose existence adds to the growing body of literature that highlights the relevance of somatic alterations that occur outside of the kinase domain. This genotype to phenotype correlation was made possible by the increasingly common use of NGS in real-time patient care, serving as a powerful example of personalized medicine in this disease.
Acknowledgments Financial support: No relevant financial support.
Author Manuscript
References 1. Ju YS, Lee W-C, Shin J-Y, Lee S, Bleazard T, Won J-K, et al. A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing. Genome Research. 2012; 22:436–45. [PubMed: 22194472] 2. Davies KD, Le AT, Theodoro MF, Skokan MC, Aisner DL, Berge EM, et al. Identifying and Targeting ROS1 Gene Fusions in Non–Small Cell Lung Cancer. Clinical Cancer Research. 2012; 18:4570–9. [PubMed: 22919003]
Cancer Discov. Author manuscript; available in PMC 2016 December 01.
Paik
Page 4
Author Manuscript Author Manuscript
3. Vaishnavi A, Capelletti M, Le AT, Kako S, Butaney M, Ercan D, et al. Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer. Nat Med. 2013; 19:1469–72. [PubMed: 24162815] 4. Wang R, Wang L, Li Y, Hu H, Shen L, Shen X, et al. FGFR1/3 Tyrosine Kinase Fusions Define a Unique Molecular Subtype of Non–Small Cell Lung Cancer. Clinical Cancer Research. 2014; 20:4107–14. [PubMed: 24850843] 5. Konduri K, Gallant J-N, Chae YK, Giles FJ, Gitlitz BJ, Gowen K, et al. EGFR fusions as novel therapeutic targets in lung cancer. Cancer Discovery. 2016 6. Conway AB, Lynch TW, Zhang Y, Fortin GS, Fung CW, Symington LS, et al. Crystal structure of a Rad51 filament. Nat Struct Mol Biol. 2004; 11:791–6. [PubMed: 15235592] 7. Ramsey JE, Daugherty MA, Kelm RJ. Hydrodynamic Studies on the Quaternary Structure of Recombinant Mouse Purβ. Journal of Biological Chemistry. 2007; 282:1552–60. [PubMed: 17121857] 8. Cho J, Pastorino S, Zeng Q, Xu X, Johnson W, Vandenberg S, et al. Glioblastoma-Derived Epidermal Growth Factor Receptor Carboxyl-Terminal Deletion Mutants Are Transforming and Are Sensitive to EGFR-Directed Therapies. Cancer Research. 2011; 71:7587–96. [PubMed: 22001862] 9. Jorissen RN, Walker F, Pouliot N, Garrett TPJ, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Experimental Cell Research. 2003; 284:31–53. [PubMed: 12648464] 10. Paik PK, Drilon A, Fan P-D, Yu H, Rekhtman N, Ginsberg MS, et al. Response to MET Inhibitors in Patients with Stage IV Lung Adenocarcinomas Harboring MET Mutations Causing Exon 14 Skipping. Cancer Discovery. 2015; 5:842–9. [PubMed: 25971939]
Author Manuscript Author Manuscript Cancer Discov. Author manuscript; available in PMC 2016 December 01.
Cancer Discov. Author manuscript; available in PMC 2016 December 01. Published in final edited form as: Cancer Discov. 2016 June ; 6(6): 574–575. doi:10.1158/2159-8290.CD-16-0478.
Something Old, Something New, Something Borrowed, Something Fused: Novel EGFR Rearrangements in Lung Adenocarcinomas Paul K. Paik1,2 1Thoracic
Author Manuscript
Oncology Service, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065
2Weill
Cornell Medical College, New York, NY 10065
Summary Mutations in the epidermal growth factor receptor (EGFR) stand as the archetype for somatic alterations that lead to oncogene addiction and that predict for response to targeted therapies. In this issue of Cancer Discovery, Konduri and colleagues report on a pair of novel oncogenic and actionable EGFR fusion events in a series of patients with lung adenocarcinomas, casting new light on this model gene.
Author Manuscript
The shift in classifying non-small cell lung cancers (NSCLC) by their molecular aberrations first and their histologies second, while not yet complete, is a reflection of the growing success of targeted therapeutics in this disease. Where histology once dictated the selection of a matched treatment (i.e. pemetrexed for non-squamous NSCLCs), it is increasingly used now as a guidepost for molecular testing: as a means to a more precise therapeutic end. This has been driven largely by the rapid pace at which actionable oncogenic events have been identified: a driver can now be found in most patients’ tumors. Over the past five years, there has also been a change in the nature of these newly identified targets as access to more comprehensive molecular testing tools has been brought into clinical use.
Author Manuscript
Hence the recent identification of a series of gene rearrangements through next-generation DNA and RNA sequencing (NGS) approaches, including those that involve rearranged during transfection (RET), c-ros oncogene 1 (ROS1), neurotrophic tyrosine kinase receptor 1 (NTRK1), and fibroblast growth factor receptors 1/3 (FGFR1/3).(1–4) These events are rare, not exceeding 1–2% per rearrangement, although they have been immediately translatable given the availability of existing tyrosine kinase inhibitors (TKIs) that have either been FDA-approved or are under investigation in other indications. Commonalities across these rearrangements have been identified, with two features that are generally present. First, each rearrangement contains the intact kinase domain of the relevant protooncogene. Second, the rearrangement partners, while varied, appear to be functionally
Contact information: Paul K. Paik, 300 East 66th Street, New York, NY 10065. 646-888-4202(p); ; Email: [email protected] Conflict of interest: The authors disclose no potential conflicts of interest.
Paik
Page 2
Author Manuscript
important, often containing coiled coil domains which can facilitate dimerization and/or localization of the fusion protein.
Author Manuscript
In this issue of Cancer Discovery, Konduri and colleagues(5) report on a pair of novel fusion events involving the archetype for targeted therapeutics in NSCLC- the epidermal growth factor receptor (EGFR)- and two 3′ partners- RAD51 and PURB- in a series of 5 patients with stage IV lung adenocarcinomas. Each of the patients’ tumors was tested in real-time with the hope of finding an actionable target using a clinically validated hybrid capturebased NGS platform (FoundationOne). Four patients had tumors harboring an EGFR fusion with a 5′ breakpoint within intron 24 (RAD51). The other harbored an EGFR fusion with a breakpoint at exon 25 (PURB). Both, consequently, had retention of an intact EGFR kinase domain. The EGFR variants were fused to either exons 4-9 of RAD51 or the 3′ untranslated region of PURB. Overall, fusion events involving EGFR exons 23-intron 25 proved to be rare, occurring in about 0.05% of more than 10,000 NSCLC tumors tested. Pre-clinical modeling of the EGFR-RAD51 fusion validated its oncogenic potential in vitro. Stable transfection of this variant in Ba/F3 cells led to IL-3 independent growth and downstream activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways to a similar extent as in Ba/F3 cells expressing EGFR L858R. EGFR-RAD51 expression in NR6 cells (an NIH 3T3 variant that lacks endogenous EGFR) was similarly transforming, leading to increased colony formation when compared to parental cells.
Author Manuscript
The authors confirmed that the 1st, 2nd, and 3rd generation EGFR TKIs erlotinib, afatinib, and osimertinib could inhibit growth and signaling in EGFR-RAD51 expressing Ba/F3 cells with low nanomolar IC50s that were not significantly different from those seen in EGFR L858R expressing cells. EGFR-RAD51 expressing cells were, however, more sensitive to growth inhibition by cetuximab than were EGFR L858R expressing cells. These in vitro data were in keeping with the durable partial responses reported in all 4 patients who were treated with erlotinib in this report (N=3 EGFR-RAD51, N=1 EGFR-PURB). The therapeutic potential of this work alone is important for its immediate clinical relevance.
Author Manuscript
As intriguing, however, are the structure-function questions that the fusion events bring to light. RAD51 is a eukaryotic homologous recombinase that self-assembles into filaments containing several RAD51 protomers.(6) Purβ (encoded by PURB) is a member of a family of ssDNA and RNA binding proteins and functions as a transcriptional repressor in vascular smooth muscle cells and myofibroblasts.(7) Prior studies have demonstrated that Purβ can also self-associate into homodimeric complexes.(7) This raises the possibility that both RAD51 and Purβ could facilitate EGFR dimerization, which would be in keeping with the presumed function of the non-kinase partners in other oncogene rearrangements. To test this hypothesis, the authors performed in silico modeling of EGFR-RAD51 based on existing structural data and showed that asymmetric dimerization of EGFR is at least geometrically possible. Whether dimerization does indeed occur in practice in either event is currently unknown.
Cancer Discov. Author manuscript; available in PMC 2016 December 01.
Paik
Page 3
Author Manuscript Author Manuscript
It is worth highlighting in light of this that EGFR C-terminal domain (CTD, spanning exons 25-28) deletions have been previously identified in glioblastomas and lung adenocarcinomas.(8) This domain is effectively deleted in the EGFR-RAD51 and EGFRPURB fusion events. CTD deletions vary in extent, from truncations of the entire CTD to partial deletions of it. At least three of these variants- an exon 27 deletion, exon 25-27 deletion, and exon 25-28 deletion- are transforming, as previously shown by Cho and colleagues.(8) In addition, these CTD variants sensitize cells to growth inhibition with erlotinib or cetuximab. Intriguingly, Cho and colleagues had also identified a novel CTD variant in an EGFR wild-type infected Ba/F3 clone that unexpectedly exhibited IL-3 independent growth. This variant was marked by an intragenic deletion of residues encoding amino acids 1,010 to 1,152 (referred to as CT Del1). Re-expression of the CT Del1 variant into parental Ba/F3 cells and NIH-3T3 cells was transforming. An orthotopic LN443 glioblastoma xenograft model stably expressing CT Del1 also generated brain tumors whose growth could be inhibited by cetuximab.
Author Manuscript
The CT Del1 variant is intriguing because of its focal nature. The deletion includes tyrosine 1045, which serves as the main binding site for the Cbl ubiquitin ligase that targets EGFR for degradation. Consistent with deletion of Y1045, Konduri and colleagues found that the EGFR-RAD51 fusion exhibited decreased EGF receptor turnover. As the authors noted, the CTD also contains a number of auto- and trans-phosphorylated docking sites for adapters that can potentiate EGFR signaling, though these are not necessary for activation of the receptor.(9) Taken together with other experimental data, the work by Konduri and colleagues suggests that the EGFR-RAD51/PURB fusions might increase EGFR activity through two mechanisms- facilitation of asymmetric dimerization and/or impairment of receptor turnover. The latter is the presumptive mechanism of action for the recently identified oncogenic exon 14 deletion in the mesenchymal epithelial transition factor (MET) proto-oncogene, which eliminates the Y1003 Cbl binding site and leads to receptor stabilization, providing a proof-of-principle for this concept.(10) In summary, Konduri and colleagues have identified novel, actionable, and rare EGFR fusions whose existence adds to the growing body of literature that highlights the relevance of somatic alterations that occur outside of the kinase domain. This genotype to phenotype correlation was made possible by the increasingly common use of NGS in real-time patient care, serving as a powerful example of personalized medicine in this disease.
Acknowledgments Financial support: No relevant financial support.
Author Manuscript
References 1. Ju YS, Lee W-C, Shin J-Y, Lee S, Bleazard T, Won J-K, et al. A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing. Genome Research. 2012; 22:436–45. [PubMed: 22194472] 2. Davies KD, Le AT, Theodoro MF, Skokan MC, Aisner DL, Berge EM, et al. Identifying and Targeting ROS1 Gene Fusions in Non–Small Cell Lung Cancer. Clinical Cancer Research. 2012; 18:4570–9. [PubMed: 22919003]
Cancer Discov. Author manuscript; available in PMC 2016 December 01.
Paik
Page 4
Author Manuscript Author Manuscript
3. Vaishnavi A, Capelletti M, Le AT, Kako S, Butaney M, Ercan D, et al. Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer. Nat Med. 2013; 19:1469–72. [PubMed: 24162815] 4. Wang R, Wang L, Li Y, Hu H, Shen L, Shen X, et al. FGFR1/3 Tyrosine Kinase Fusions Define a Unique Molecular Subtype of Non–Small Cell Lung Cancer. Clinical Cancer Research. 2014; 20:4107–14. [PubMed: 24850843] 5. Konduri K, Gallant J-N, Chae YK, Giles FJ, Gitlitz BJ, Gowen K, et al. EGFR fusions as novel therapeutic targets in lung cancer. Cancer Discovery. 2016 6. Conway AB, Lynch TW, Zhang Y, Fortin GS, Fung CW, Symington LS, et al. Crystal structure of a Rad51 filament. Nat Struct Mol Biol. 2004; 11:791–6. [PubMed: 15235592] 7. Ramsey JE, Daugherty MA, Kelm RJ. Hydrodynamic Studies on the Quaternary Structure of Recombinant Mouse Purβ. Journal of Biological Chemistry. 2007; 282:1552–60. [PubMed: 17121857] 8. Cho J, Pastorino S, Zeng Q, Xu X, Johnson W, Vandenberg S, et al. Glioblastoma-Derived Epidermal Growth Factor Receptor Carboxyl-Terminal Deletion Mutants Are Transforming and Are Sensitive to EGFR-Directed Therapies. Cancer Research. 2011; 71:7587–96. [PubMed: 22001862] 9. Jorissen RN, Walker F, Pouliot N, Garrett TPJ, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Experimental Cell Research. 2003; 284:31–53. [PubMed: 12648464] 10. Paik PK, Drilon A, Fan P-D, Yu H, Rekhtman N, Ginsberg MS, et al. Response to MET Inhibitors in Patients with Stage IV Lung Adenocarcinomas Harboring MET Mutations Causing Exon 14 Skipping. Cancer Discovery. 2015; 5:842–9. [PubMed: 25971939]
Author Manuscript Author Manuscript Cancer Discov. Author manuscript; available in PMC 2016 December 01.
