Is NRG2α Fusion a “Doppelgänger” to NRG1α/β Fusions in Oncology?
Authors: Misako Nagasaka, Sai-Hong Ignatius Ou
Journal: Journal of Thoracic Oncology (2020)

Neuregulins (NRGs) are cellular signaling proteins that contain epidermal growth factor (EGF)–like domains and play important roles in the development of the nervous and cardiovascular systems. NRG1 and NRG2 are members of six distinct NRG genes (NRG1, NRG2, NRG3, NRG4, NRG5, [tomoregulin-1, transmembrane protein with EGF like and two follistatin like domains 1 {TMEFF1}] and NRG6 [Neuroglycan-C, chondroitin sulfate proteoglycan 5 {CSPG5}, chicken acidic leucine-rich EGF-like domain-containing brain protein {CALEB}) that share an EGF-like domain.1Mei L. Nave K.A. Neuregulin-ERBB signaling in the nervous system and neuropsychiatric diseases.Neuron. 2015; 83: 27-49Abstract Full Text Full Text PDF Scopus (368) Google Scholar The EGF-like domain is generated by alternative exon splicing, in which a core EGF-like domain exon is spliced together with an exon encoding either the alpha-amino acid (α-isoform) sequence or the beta-amino acid (β-isoform) sequence to generate the full-length EGF-like domain.2Falls D.L. Neuregulins: functions, forms, and signaling strategies.Exp Cell Res. 2003; 284: 14-30Crossref PubMed Scopus (852) Google Scholar In this issue, Kohsaka et al.3Kohsaka S. Hayashi T. Nagano M. et al.Identification of novel CD74-NRG2α fusion from comprehensive profiling of lung adenocarcinoma in Japanese never or light smokers.J Thorac Oncol. 2020; 15: 948-961Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar performed whole transcriptome sequencing in 125 Asian light-smokers/never-smokers with “driver-negative” adenocarcinoma of the lung. Among the genetic alterations identified with whole transcriptome sequencing were three CD74-NRG1α/β fusions and a novel CD74-NRG2α fusion.3Kohsaka S. Hayashi T. Nagano M. et al.Identification of novel CD74-NRG2α fusion from comprehensive profiling of lung adenocarcinoma in Japanese never or light smokers.J Thorac Oncol. 2020; 15: 948-961Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Of note, no NTRK fusions were detected using whole transcriptome sequencing, indicating the even lower incidence of NTRK fusions in adenocarcinoma of the lung than NRG fusions. The histology of the CD74-NRG2α fusion–positive tumor was acinar adenocarcinoma rather than the invasive mucinous adenocarcinoma histology among all three CD74-NRG1α/β samples. Although there is now amble evidence from case reports that NRG1 fusions are actionable driver mutations, especially in NSCLC and pancreatic ductal adenocarcinoma,4Drilon A. Somwar R. Mangatt B.P. et al.Response to ERBB3-directed targeted therapy in NRG1-rearranged cancers.Cancer Discov. 2018; 8: 686-695Crossref PubMed Scopus (106) Google Scholar, 5Nagasaka M. Ou S.I. Neuregulin 1 fusion-positive NSCLC.J Thorac Oncol. 2019; 14: 1354-1359Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 6Heining C. Horak P. Uhrig S. et al.NRG1 fusions in KRAS wild-type pancreatic cancer.Cancer Discov. 2018; 8: 1087-1095Crossref PubMed Scopus (123) Google Scholar, 7Jones M.R. Williamson L.M. Topham J.T. et al.NRG1 gene fusions are recurrent, clinically actionable gene rearrangements in KRAS wild-type pancreatic ductal adenocarcinoma.Clin Cancer Res. 2019; 25: 4674-4681Crossref PubMed Scopus (83) Google Scholar several lines of evidence caution us to immediately conclude that NRG2α fusions should automatically be “anointed” as an oncogenic driver mutation based just on its homology in the EGF-like domain and similar spectrum of binding to HER2/HER3 and HER2/HER4. First, both NRG1 and NRG2 bind to HER3 and HER4, and the binding specificity within each NRG is largely determined by the α- and β-isoforms. To state it simply, the EGF-like α-isoform has generally lower affinity for HER2 members than its corresponding β-isoform.8Jones J.T. Akita R.W. Sliwkowski M.X. Binding specificities and affinities of egf domains for ErbB receptors.FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (320) Google Scholar,9Pinkas-Kramarski R. Shelly M. Guarino B.C. et al.ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network.Mol Cell Biol. 1998; 18: 6090-6101Crossref PubMed Scopus (127) Google Scholar In vitro experiments have revealed that NRG1β binds with higher affinity to HER2/HER3 than HER2/HER4, whereas the reverse is true for NRG2β.8Jones J.T. Akita R.W. Sliwkowski M.X. Binding specificities and affinities of egf domains for ErbB receptors.FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (320) Google Scholar,9Pinkas-Kramarski R. Shelly M. Guarino B.C. et al.ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network.Mol Cell Biol. 1998; 18: 6090-6101Crossref PubMed Scopus (127) Google Scholar Importantly, in one study, NRG2α was found to bind with high affinity to HER4 monomers/dimers, with low affinity to HER2/HER4, and with only minimal affinity to HER2/HER3.8Jones J.T. Akita R.W. Sliwkowski M.X. Binding specificities and affinities of egf domains for ErbB receptors.FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (320) Google Scholar Thus, it is not surprising that Kohsaka et al.3Kohsaka S. Hayashi T. Nagano M. et al.Identification of novel CD74-NRG2α fusion from comprehensive profiling of lung adenocarcinoma in Japanese never or light smokers.J Thorac Oncol. 2020; 15: 948-961Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar only identified moderate HER4 but no HER3 phosphorylation by immunohistochemistry (IHC) in the CD74-NRG2α fusion sample. Furthermore, there was no phosphorylation of HER2 or EGFR by IHC in the CD74-NRG2α fusion sample. In contrast, CD74-NRG1α/β–positive NSCLC samples were positive not just for HER3 phosphorylation but also for phosphorylation in EGFR, HER2, and HER4. IHC for p-HER3 has been used to detect NRG1 fusions with high sensitivity and specificity.10Fernandez-Cuesta L. Plenker D. Osada H. et al.CD74-NRG1 fusions in lung adenocarcinoma.Cancer Discov. 2014; 4: 415-422Crossref PubMed Scopus (192) Google Scholar,11Trombetta D. Graziano P. Scarpa A. et al.Frequent NRG1 fusions in Caucasian pulmonary mucinous adenocarcinoma predicted by phospho-ErbB3 expression.Oncotarget. 2018; 9: 9661-9671Crossref PubMed Scopus (32) Google Scholar If indeed NRG2α fusion turns out to be the oncogenic driver in this one CD74-NRG2α fusion–positive tumor, pairwise screening for p-HER3/p-HER4 may potentially serve as a screening method for NRG1 and NRG2 fusions. Second, NRG1 knockout mice generated by disrupting either the NRG1 core EGF-like domain or NRG1β immunoglobulin-like domain were embryonically lethal owing to substantial maldevelopment of the heart and the nervous system.12Meyer D. Birchmeier C. Multiple essential functions of neuregulin in development.Nature. 1995; 378: 386-390Crossref PubMed Scopus (1049) Google Scholar,13Kramer R. Bucay N. Kane D.J. Martin L.E. Tarpley J.E. Theill L.E. Neuregulins with an Ig-like domain are essential for mouse myocardial and neuronal development.Proc Natl Acad Sci U S A. 1996; 93: 4833-4838Crossref PubMed Scopus (190) Google Scholar In contrast, NRG1α-specific knockout mice survived to adulthood with only pronounced defects in mammary gland lobuloalveolar development.14Li L. Cleary S. Mandarano M.A. Long W. Birchmeier C. Jones F.E. The breast proto-oncogene, HRGalpha regulates epithelial proliferation and lobuloalveolar development in the mouse mammary gland.Oncogene. 2002; 21: 4900-4907Crossref PubMed Scopus (78) Google Scholar Furthermore, NRG2β knockout mice generated by disrupting both the NRG2 immunoglobulin-like and core EGF-like domains were viable and lived to 18 months with the only exception of delayed weight gain in the early weeks.15Britto J.M. Lukehurst S. Weller R. et al.Generation and characterization of neuregulin-2-deficient mice.Mol Cell Biol. 2004; 24: 8221-8226Crossref PubMed Scopus (35) Google Scholar Taken together, although NRG2 has normal function in humans, its functions seem largely subservient to NRG1; the β-isoforms of NRG1 and NRG2 in general may be the more vital isoforms than the α-isoforms. Finally, binding of NRG1 and NRG2 to HER2/HER3 activates different phosphorylation sites in HER2 and HER3, leading to recruitment of different sets of subcellular signaling proteins and signaling pathway and prolonged activation.16Crovello C.S. Lai C. Cantley L.C. Carraway 3rd, K.L. Differential signaling by the epidermal growth factor-like growth factors neuregulin-1 and neuregulin-2.J Biol Chem. 1998; 273: 26954-26961Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 17Sweeny C. Lai C. Riese 2nd, D.J. Diamonti A.J. Cantley L.C. Carraway 3rd, K.L. Ligand discrimination in signaling through an Erb4 receptor homodimer.J Biol Chem. 2000; 275: 19803-19807Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18Sweeny C. Fambrough D. Huard C. et al.Growth factor specific signaling pathway stimulation and gene expression mediated by ErB receptors.J Biol Chem. 2001; 276: 22685-22698Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Thus, activation of the HER3 pathway by NRG1 or NRG2 in vitro may not lead to the same phenotypic outcome adding to the complexity of NRG signaling. In addition, this report by Kohsaka et al.3Kohsaka S. Hayashi T. Nagano M. et al.Identification of novel CD74-NRG2α fusion from comprehensive profiling of lung adenocarcinoma in Japanese never or light smokers.J Thorac Oncol. 2020; 15: 948-961Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar adds important findings to the field of NRG1 fusions. Whereas the proportion of α- to β-isoforms of normal NRG1 or in the oncogenic NRG1 fusions remains unknown in the literature, both CD74-NRG1α and CD74-NRG1β variants were expressed in varying degrees in the three samples identified (NRG1β/NRG1α ratios were 1.15, 0.76, and 0.38 among the three NRG1α/β fusions reported).3Kohsaka S. Hayashi T. Nagano M. et al.Identification of novel CD74-NRG2α fusion from comprehensive profiling of lung adenocarcinoma in Japanese never or light smokers.J Thorac Oncol. 2020; 15: 948-961Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Together with the discovery of the CD74-NRG2α fusion, it will now be important to identify the exact isoform (and the ratio) of the EGF-like domain of the NRG1 and NRG2 fusions in malignancies, given the difference in binding specificities and affinities to the HER family members. Given also the ever-expanding fusion partners being identified in the NRG1 fusion,4Drilon A. Somwar R. Mangatt B.P. et al.Response to ERBB3-directed targeted therapy in NRG1-rearranged cancers.Cancer Discov. 2018; 8: 686-695Crossref PubMed Scopus (106) Google Scholar, 5Nagasaka M. Ou S.I. Neuregulin 1 fusion-positive NSCLC.J Thorac Oncol. 2019; 14: 1354-1359Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 6Heining C. Horak P. Uhrig S. et al.NRG1 fusions in KRAS wild-type pancreatic cancer.Cancer Discov. 2018; 8: 1087-1095Crossref PubMed Scopus (123) Google Scholar,19Jonna S. Feldman R.A. Swensen J. et al.Detection of NRG1 gene fusions in solid tumors.Clin Cancer Res. 2019; 25: 4966-4972Crossref PubMed Scopus (98) Google Scholar it is not farfetch to speculate that the β-to-α ratio may be determined by the fusion partners and/or the fusion breakpoint in NRG1. This will have implication in the methods to detect NRG fusions as methods that do not sequence the full EGF-binding domain and its neighboring region or fusion partners (to differentiate from endogeneous NRGs from oncogenic NRG fusions) may not be able to differentiate the α-isoform from the β-isoform and may not provide all the important biological data to optimally target NRG fusions in oncology. Much work remains to be done to understand if HER4 phosphorylation alone by NRG2α fusion may promote oncogenesis and if so, how. Some of the current clinical trials targeting NRG1 fusions, such as zenocutuzumab (MCLA-128) (HER2/HER3 bi-specific antibody) (ClinicalTrials.gov: NCT02912949), are specific for the HER3 pathway and may not be effective against NRG2α if NRG2α activates through HER4 but not HER2/HER3 as suggested by the phospho-IHC findings by Kohsaka et al.3Kohsaka S. Hayashi T. Nagano M. et al.Identification of novel CD74-NRG2α fusion from comprehensive profiling of lung adenocarcinoma in Japanese never or light smokers.J Thorac Oncol. 2020; 15: 948-961Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Tarloxotinib, a hypoxia-inducible pan-HER2 inhibitor, is also being investigated against NRG1 fusions and this may be better suited to target NRG2α fusions (ClinicalTrials.gov: NCT03805841). Similarly, another pan-HER inhibitor, afatinib, is being investigated against NRG1 fusions as part of the TAPUR trial (group 18, NRG1 fusion) that may also target NRG2α fusions (ClinicalTrials.gov: NCT02693535). “Doppelgänger” is a term used to describe people whose appearances are so similar that they can be mistaken as twins but are genetically completely unrelated. Although NRG1α/β and NRG2α share amino acid homology among the EGF-like domains, it remains to be determined whether the identified CD74-NRG2α fusion is oncogenic or not. In addition, will oncogenic NRG2β fusions be identified soon in NSCLC and NRG2α fusions other solid malignancies? The discovery of the CD74-NRG2α fusion in NSCLC by Kohsaka et al.3Kohsaka S. Hayashi T. Nagano M. et al.Identification of novel CD74-NRG2α fusion from comprehensive profiling of lung adenocarcinoma in Japanese never or light smokers.J Thorac Oncol. 2020; 15: 948-961Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar will surely prompt immediate endeavors to search for NRG2 fusions in NSCLC and other solid tumors. Direct comparison between CD74-NRG2α (and likely CD74-NRG2β) xenograft to CD74-NRG1α and CD74-NRG1β xenograft tumorigenesis modeling will need to be performed to dissect the potency of each oncogenic NRG fusion (NRG1α, NRG1β, NRG2α, NRG2β) similar to the elegant experiments performed by Professor Mano's group.20Soda M. Choi Y.L. Enomoto M. et al.Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4322) Google Scholar for EML4-ALK fusion more than a decade ago. We may well be witnessing the dawn of an era of “ligand fusion” in solid tumors after the past decade of “receptor tyrosine kinase fusion” in NSCLC and other solid tumors.21Schram A.M. Chang M.T. Jonsson P. Drilon A. Fusions in solid tumours: diagnostic strategies, targeted therapy, and acquired resistance.Nat Rev Clin Oncol. 2017; 14: 735-748Crossref PubMed Scopus (188) Google Scholar Identification of Novel CD74-NRG2α Fusion From Comprehensive Profiling of Lung Adenocarcinoma in Japanese Never or Light SmokersJournal of Thoracic OncologyVol. 15Issue 6PreviewStudies are yet to characterize the differences in molecular profiles of lung adenocarcinoma (LUAD) among divergent ethnic groups. Herein, we conducted comprehensive molecular profiling of LUAD in never or light smokers from Asia to discover novel targetable mutations and prognostic biomarkers of this distinct disease entity. Full-Text PDF Open Access